Novel cancer-related roles of GCN2

Novel cancer-related roles of GCN2

Prabin Sharma Humagain

Master Thesis in

Molecular Biology and Biochemistry 60 credits

Department of Biosciences

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

June / 2020

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Novel cancer-related roles of GCN2

Prabin Sharma Humagain

Master Thesis in Molecular Biology and Biochemistry 60 credits

Department of Biosciences

Faculty of Mathematics and Natural Sciences UNIVERSITY OF OSLO

June / 2020

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© Prabin Sharma Humagain 2020

Novel cancer related roles of GCN2 Supervisor: Beata Grallert

http://www.duo.uio.no/

Trykk: Reprosentralen, Universitetet i Oslo

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Abstract

GCN2, an amino-acid deprivation sensor is one of the kinases activated during the integrated stress response. Its role in normal as well as in cancer cells during stress induced by starvation has garnered a lot of attention. Most of the literature describes GCN2’s role based on its action on the eIF2-ATF4 pathway. However, recent findings suggest that this may not be the only way that GCN2 acts and may not be the only reason why it is important in the context of cancer. Therefore, our group began a study of transcriptomic data from cervical cancer patients to analyze the correlation of various genes with GCN2 and found different functional groups associated with the genes that showed correlation with GCN2. So, in this study, we performed correlation analyses for selected groups of genes using different cell lines. We found that there is a correlation between GCN2 and genes associated with cell motility, mitosis, oxidative phosphorylation, and immune response. Based on these results, phenotypic analyses were done. We were also able to show through phenotypic analysis that GCN2 has a role in directional migration while our preliminary result also suggests an increase in mitochondrial respiration in the case of GCN2 depletion. This study has been successful in verifying the correlation of GCN2 with some genes that are important in cancer survival and was also able to prove the role of GCN2 in pathways other than the ISR relevant to cancer. Further

investigation of the findings of this study could prove valuable in understanding the role of GCN2 and using it as a target in therapy against cancer.

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Acknowledgements

The project presented in this thesis was carried out at the Department of Radiation Biology, Institute for Cancer Research at the Norwegian Radium Hospital, Oslo University Hospital.

First and foremost, I want to express my gratitude to my main supervisor, Beáta Grallert for having me as her master student and providing an opportunity to work in this really interesting project. Your enthusiasm during our discussions was contagious, which helped me get excited and stay stress-free during this work. Those discussions also helped me to not only understand things but also improved upon my critical thinking. Your help during the lab work and the writing process were invaluable for me.

I would also like to express deep gratitude to Theodossis Athanassios Theodossiou, my co-supervisor.

Your assistance in the investigation of the cellular respiration with SeaHorse was of great

importance. More than that, your constant cheerful and welcoming attitude towards everyone was always a welcome sight.

Furthermore, I am particularly grateful for the assistance provided by Carmen Herrera from the Cancer Molecular medicine group (Jorrit Enserink’s group) for the study of gene expression in qPCR.

Your guidance throughout the whole process was meticulous and your insight into the method very valuable.

I wish to thank Lilian Lindbergsengen for her help and advice in the everyday laboratory work, especially with western blots.

I would also like to extend special thanks to my fellow master student, Ingrid Wilhelmsen for her help and encouragement during the writing process while our conversation on supposedly not so popular opinions was always fun.

Finally, I want to thank my friends and family for their support and encouragement during these two years of study, away from home. I could not have done it without them.

-Prabin Sharma Humagain, June, 2020

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Table of Contents

Abstract ... V Acknowledgements ... VII

1. Introduction ... 1

Aims of the study ... 8

2. METHODS ... 9

2.1 Cell Culture Methods ... 9

2.2 Cell count ... 12

2.2.1 Automated counting ... 12

2.2.2 Manual counting ... 13

2.3 Transient transfection ... 14

2.4 Investigation of Protein Expression ... 15

2.4.1 Protein extraction ... 16

2.4.2 Protein measurement... 17

2.4.3 Protein separation ... 18

2.4.4 Protein transfer ... 19

2.4.5 Immunodetection ... 19

2.5 Analysis of gene expression ... 20

2.5.1 RNA isolation from cells ... 21

2.5.2 RNA measurement ... 23

2.5.3 Reverse transcription ... 23

2.5.4 Testing the product of reverse transcription ... 24

2.5.5 Real-time quantitative PCR ... 26

2.6 Functionality of the transduced GCN2 ... 28

2.6.1.1 UV irradiation of the cells ... 28

2.6.1.2 Cell lysis and collection ... 29

2.6.1.3 Immunoprecipitation... 29

2.6.1.4 Kinase Assay ... 30

2.6.1.5 Phos-tag gel electrophoresis ... 31

2.7 Migration Assay ... 33

2.7.1 Directional migration ... 33

2.7.2 Random movement of cells ... 35

2.8 Cellular Respiration ... 36

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2.9 Statistical analysis ... 39

3. RESULTS ... 41

3.1 Selecting the gene of interest for analysis. ... 41

3.2Testing the level of GCN2 expression in the different cell lines to be used. ... 45

3.3 Kinase activity test of the transduced gene in HeLa cells. ... 46

3.3.1 In-vitro kinase activity of transduced GCN2 ... 46

3.3.2 Testing in-vivo kinase activity of the transduced GCN2 ... 47

3.4 Assessing the correlation of selected genes to GCN2 at transcript level... 48

3.4.1 Testing primers for their suitability in qPCR ... 49

3.4.2 Selection of housekeeping gene ... 51

3.4.3 Analyzing the correlation of genes of interest with GCN2 overexpression in HeLa cells. ... 52

3.4.4 Determining the effect of number of generations in correlation of gene of interest with GCN2. ... 53

3.4.5 Selection of housekeeping gene for RPE cells ... 54

3.4.6 Analysis of correlation of genes of interest with GCN2 in case of overexpression and knockdown in HeLa cells. ... 55

3.4.7 Analysis of correlation of genes of interest with GCN2 in case of overexpression and knockdown in RPE cells. ... 56

3.4.8 Testing the effect of the number of passages in GCN2 expression ... 57

3.4.9 Analysis of the effect of ATF4 knockdown, individually and with GCN2 in RPE cells. ... 58

3.5 Test for effect of GCN2 level in different phenotypes in cells ... 59

3.5.1 Evaluating the effect of GCN2 levels on cellular movement ... 59

3.5.2 Evaluation of the effect of GCN2 expression on cellular metabolism. ... 67

3.5.3 Evaluation of the effect of GCN2 levels on cell division ... 69

4. Discussion ... 70

4.1 Challenges and limitations ... 70

4.2 GCN2 has a role in cell motility... 72

4.3 GCN2 overexpression decreases cellular respiration ... 75

4.4 GCN2 depletion delays the prometaphase to anaphase progression ... 77

4.5 GCN2 showed correlation with genes associated with Immune response. ... 79

4.6 Further experimentation required to determine the ATF4 dependence of the

effects of GCN2 on genes of interest. ... 80

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Conclusion ... 81

Appendix A: Abbreviations ... 82

Appendix B: Cell lines, Materials, Instruments, Reagents, Buffers and Softwares. ... 85

Appendix C: Different buffers and their composition ... 90

Appendix D: Primer and siRNA Sequence... 92

Appendix E: Supplementary data ... 93

References ... 99

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1. Introduction

Cancer is a rapid, uncontrolled growth of cells. This growth can affect the organism in multiple ways.

Based on the organ or tissue involved in cancer development, and the aggressiveness of cancer, it can cause a wide range of effects. It can cause physiologic changes in the body like hormonal imbalance in case of thyroid cancer. Or it can cause the failure of the body’s essential systems by its overgrowth in those body parts like the respiratory system or the digestive system and so on [1]. One of the models that is widely accepted as being responsible for the rise of cancer is the many-

mutation model [2]. This model states that for the development of a cancer, there must be loss or impairment of many genes affecting different functions of cells including the regulation of the cell division. Also, for multiple mutations to occur, it is hypothesized that the initial mutation should be in those genes that prevent mutations like p53 or Retinoblastoma (Rb) genes, also known as tumor suppressor genes, which are involved in deoxyribonucleic acid (DNA) damage repair, inhibition of cell division, suppression of metastasis and induction of apoptosis [3]. This would result in the

accumulation of mutations and impairment in various regulatory functions resulting in rapid cell growth. The major characteristics the cells need to gain to be classified as cancer cells are known as the hallmarks of cancer. The major hallmarks of cancer are shown in Figure 1 [4, 5].

Figure 1 Hallmarks of cancer. Image retrieved from wordpress.com

Continued cell division of cancer cells reflects four of the hallmarks of cancer cells; sustaining proliferation signal, evading growth suppression, resisting cell death, and enabling replicative immortality. As stated earlier, one of the ways this is achieved is by alteration of various proteins

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involved in maintaining proper cell division [5, 6]. These alterations can render some proteins non- functional like tumour suppressor genes (TSG). TSGs are responsible for repressing the progression of the cell cycle through proteins that are necessary for progression in cases of irregularities in DNA mutations during DNA replication. Other proteins that may lose their function are checkpoint proteins that make sure of the chromosomal segregation before proceeding to the next step in the cell cycle. However, other genes get abnormally activated and are known as oncogenes, which results in growth deregulation [7]. Oncogenes are necessary for proper function and their activity is regulated but in cancer, this regulation is impaired and the oncogenes are overexpressed or are in a constitutively active state. In some other cases, it also includes a gain of new functions like activation of telomerase enzyme which is seen in almost 90% of the cancers and is responsible to prevent the limitation on number of cell divisions a cell can go through [8, 9].

Similarly, alteration in proteins involved in cytoskeleton arrangement and signal transduction can play a huge role in activating invasion and metastasis, another hallmark of cancer [10, 11], which helps spread cancer to different parts of the body and is a major factor in cancer mortality [12].

However, this is a case of cancer cells taking benefit of normal cellular processes, as normal cells need to move about for basic biological processes. For example, random movement of cells is

associated with cell-cell interaction, cell adhesion, and signal transfer between the cells. On the other hand, directional movement of cells, especially immune cells towards the chemokines and cytokines is a vital process of our immune response against pathogens. Not just the directional movement, but the random movement of the cells is also found to require internal signal transduction and changes in this signal transduction process can bring about change in migratory potential [13]. This kind of movement can be important for cancer cells not only to migrate to a favourable environment but also to evade the immune system.

Other properties gained by cancer cells for survival include changes in the metabolic mechanism for energy. In the cancer microenvironment, where the supply of oxygen can be limited, along with inducing angiogenesis, cancer cells also tend to undergo a metabolic shift from oxidative

phosphorylation and towards glycolysis for Adenosine triphosphate (ATP) production, termed the Warburg effect [14]. This not only helps provide energy but along with the Krebs’s cycle, also provides precursors for the high anabolic demands of cancer cells [15].

Finally, for cancer cells to survive, they need to have mechanisms to prevent detection and

destruction by the immune systems of the body. Since both innate and adaptive immune system can kill the cancer cells [16], the cancer cells can develop a variety of methods to evade the immune

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system. This can involve lack of tumour-antigen recognition, resistance to cell death, or induction of immunological ignorance and tolerance [17].

Normal cells also exhibit changes but in a milder form to negate the imbalance in their surroundings, in a process termed homeostasis. Homeostasis refers to the tendency to maintain a relatively stable physiological condition required for survival [18]. Homeostasis is required for the survival and functioning of the cells. The changes experienced by the cells can be on various aspects as

exemplified before like nutrients, changes in physical conditions like pH or osmolarity. It can also be exposure to foreign substances like different chemicals, pathogens, or exposure to radiation. All of these cause stress to the cells which elicit responses, which cells have acquired to cope against these kinds of stress [19].

One of the methods developed by cells to cope against these stresses is known as the Integrated Stress Response (ISR). ISR is an intricate, adaptive, pro-survival but short-lived pathway in eukaryotes that allows cells to identify the loss of homeostasis and adapt in a way to rectify the imbalance [20].

It can be activated by various factors including amino-acid starvation, heme deficiency, endoplasmic reticulum (ER) stress caused by misfolded protein, ultraviolet (UV) irradiation, or viral infection resulting in double-stranded Ribonucleic Acid (dsRNA) in the bloodstream [21]. These stresses are sensed by specific kinases namely, General control nonderepressible 2 (GCN2), Heme-regulated inhibitor (HRI), Protein kinase, interferon-inducible dsRNA-activated (PKR), and PKR-like Endoplasmic Reticulum Kinase (PERK).

These kinases when activated phosphorylate eukaryotic Initiation Factor 2-α (eIF2-α) at serine-51 which finally causes upregulation of translation of the selected mRNAs while causing global

translational arrest [21, 22]. This effect has been described as the result of the eIF2α-ATF4 pathway [20]. Before describing the eIF2α-ATF4 pathway, we need to understand how eIF2 is involved in translation. As shown in Figure 2A, eIF2 is a non-phosphorylated complex with bound Guanosine diphosphate (GDP). Under normal conditions, GDP would be exchanged by Guanosine triphosphate (GTP) followed by binding of transfer RNA (tRNA) that is bound to methionine to form the ternary complex. This complex will go on to bind with the ribosome and initiate the translation [23] while GTP gets hydrolyzed to form GDP.

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A. B

Figure 2 Role of eIF2 in general translation. A) Formation of Ternary complex in general initiation of translation. B) Effect of eIF2 phosphorylation in general translation [24].

Under the condition of stress (Fig. 2B), various kinases get activated based on the nature of the stress and phosphorylate eIF2 at serine-51 residue of the α subunit. In this phosphorylated form, eIF2 is unable to exchange GDP for GTP which leads to an inability to form the ternary complex. The overall result of this is the downregulation of global protein synthesis and the selective translation of specific genes adaptive to stress [23]. The mechanism of translation of specific messenger RNAs (mRNAs) under stress conditions is not well-understood. Especially, among the mRNAs whose translation is initiated by ternary complex, only Activating transcription factor 4 (ATF4), a transcription factor, is identified and there are hypotheses on how ATF4 is affected. However, the identity and mechanism of effect on other mRNAs affected by the ternary complex are not known.

The widely accepted hypothesis for induction and translation of ATF4 is called delayed reinitiation model (Fig. 3) [25]. According to this model, the mRNA of ATF4 has two upstream open reading frames (uORF) in the 5’ end (Fig. 3). ORF is the continuous segment of a reading frame that can be translated, starts with a start codon and ends in a stop codon [26]. uORFs are small ORFs found upstream of the primary ORF and are generally responsible for the regulation of translation of the primary ORF [27, 28].

Under normal conditions, when there is a high concentration of the eIF2-GTP, ternary complexes are readily formed. This can go on and bind to the scanning 40s ribosome when an open reading frame is encountered. In case of ATF4, when the translation of uORF-1 is completed, other components of the initiation complex except the 40s scanning ribosomal subunit dissociate. The 40s ribosome continues scanning and reaches uORF-2 and initiates the translation. However, since the start codon of the ATF4 is out-of-frame within the uORF-2, ATF4 is not translated [25].

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Figure 3 Mechanism of action in the translation of ATF4 according to delayed reinitiation model [29].

The explanation of the figure can be found in the text. Red bars represent uORF-1 and uORF-2. Blue bar represents ATF4-ORF. The ribosome is shown as purple oval structures with newly formed polypeptide chain.

In the case of stress when eIF2 is phosphorylated and the concentration of ternary complex is low, it affects the translation of uORF-2. This is because even when the 40s ribosome encounters the start codon for uORF-2, because of the lack of availability of ternary complex, translation cannot be initiated. So, the scanning ribosome keeps moving in the 3’ direction and encounters the start codon of ATF4 and the translation of the ATF4 can begin [25]. Then ATF4 can regulate different genes important for cell survival specifically related to metabolic adaption. These changes are targeted to alleviate the effect caused by the stress which would otherwise be fatal to the cell. These changes try to optimize the processes in the cells and help the cell survive the stress. And since the known method of ATF4-mRNA translation is through eIF2α phosphorylation, it is generally tested and discussed.

ISR is of particular importance for cancer cells because of the already discussed stress they

experience as well as their competition for nutrients with normal cells, which is exacerbated by their high anabolic demand. Especially chronic hypoxia and nutrient deprivation are observed in tumours because of the abnormal vascularization [30]. Recently, there has been a lot of research in

understanding the impact of ISR in cancer cells and it has been shown that ISR is important for tumour growth [31, 32]. This has encouraged researchers to target ISR for cancer treatment. Some of the methods being looked upon are inhibition of ATF4 expression [33] or the use of ISR inhibitor (ISRIB) that inhibits the downstream effect of eIF2α phosphorylation [34].

Among the four kinases involved in ISR, GCN2 is of particular interest. Our group has found a negative correlation between elevated GCN2 expression and patient survival in case of cervical

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cancers [collaboration Heidi Lyng, Christina Fjeldbo, unpublished]. This result suggests that GCN2 is important for cancer cells to thrive and cancer to be aggressive. The impact of GCN2 function is most likely similar in other cancers also as evident by the genome-wide study by Tsherniak that showed that many cancers are dependent on GCN2 (Fig 4) [32].

Moreover, the dependency of cancer on GCN2 has also been shown by a new study where the researchers found that activation of GCN2-eIF2-ATF4 pathway was necessary for proliferation of tumours overexpressing of MYC-gene [35]. MYC-gene, which is a proto-oncogene and a transcription factor, when overexpressed causes an increase in protein synthesis. This will result in high demand for amino-acids and other intermediates resulting in starvation, which in turn can activate ISR through GCN2 [35]. The same process might be used by many other cancers as well and highlights the importance of GCN2 in cancer progression.

Figure 4 Dependency scores of different cancers on GCN2. Cancers that gives dependency closer to -1 are dependent on GCN2 while those closer to 0 does not hold any correlation.

However, not all the functions of GCN2 can be explained as the result of eIF2α phosphorylation. For instance, it is also found that GCN2 level determines the sensitivity of the cancer cells toward Na⁺/K⁺- ATPase ligand-induced apoptosis [36]. It was found that Na⁺/K⁺-ATPase ligand resulted in

phosphorylation of GCN2 that prevented GCN2 degradation by proteases. This increased-GCN2-level was then responsible for increased apoptosis through post-translational mechanisms which are not well understood and may not be a described function of GCN2 in cell survival.

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There are also various ongoing studies in understanding the role of GCN2 in T-cell responses in the tumour microenvironment. The tumour microenvironment is not a suitable environment for immune surveillance because of the low nutrient or the hypoxic conditions. In normal conditions, this would elicit GCN2 response causing apoptosis of the T-cells. However, researchers have found that the use of GCN2 inhibitor in a low nutrient environment can relieve T-cell suppression and promotes effector function of the CD8 T-cells for tumour surveillance [37].

To support the hypothesis that there is more to GCN2, than phosphorylation of eIF2α, among the five domains of the GCN2 (Fig. 5), not all of them have their functions clearly defined. Curiosity in the role of GCN2 in biological processes is also increased by the notion that four different kinases are leading to the same response which is not the efficient way of resource management and does not sit in line with general evolution. Besides these, there are other functions of GCN2 that are not strictly

associated with ISR or a response to amino acid deprivation like antigen presentation [38] or regulation of synaptic plasticity [39].

Figure 5 The domain organization of GCN2. From left to right, these domains are an N-terminal RWD (RING-fingerproteins, WD repeat-containing proteins and the yeast DEAD-like helicases), a

pseudokinase domain, a kinase domain, a ‘HisRS-like’ domain and a C-terminal domain.

In addition to these arguments, a study from our group [40] showed that upon UVC irradiation of fission yeast cells, there was a delay in G1-S transition of the cell cycle. This was dependent on GCN2 and accompanied by eIF2α phosphorylation [40]. This brings the question of how GCN2 can affect the cell cycle.

This and the other facts referring to the importance of GCN2 in cancer cells survival along with many unexplained functions piqued the interest of our group in finding GCN2-dependent functions

important for cancer progression by screening cervical cancer patients’ transcriptomic data to fish out genes correlating to GCN2, which was the main subject of a previous master thesis [41]. In her thesis work, Laura Pesqueira was able to identify over 50 genes correlated with GCN2. The products of these genes were involved in various cellular functions including cell motility, oxidative

phosphorylation, DNA repair, cell cycle regulation, immune system regulation and others [41]. This opens up a possibility to understand the newer functions of GCN2 based on the functions of the genes that show correlation with GCN2. Understanding these mechanisms can be the first step in exploiting them to treat cancer.

8 Aims of the study

Our main goal is to investigate the cancer-related novel functions of GCN2.

To this end, we wish to

 Verify the correlation observed in the patients’ data between GCN2 and other genes.

Approach: Use cell lines with GCN2 overexpression or depletion to observe the difference in transcript levels of the chosen genes of interest using qPCR.

 Evaluate phenotypic consequences of depleting or overexpressing GCN2 in mechanisms suggested by pathway analyses of genes correlating with GCN2 in the biobank.

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2. METHODS

2.1 Cell Culture Methods

Cell-culture is the process of growing cells in a controlled environment. It requires maintenance of the controlled conditions as close to the natural environment as possible. Maintenance of this condition requires a supply of the nutrients, growth factor, hormones, chemical buffers, antibiotics, and proper management of physical conditions like temperature, and gas composition. Since cell culture is a simple but effective model for the investigation of the different properties of cells, reliably studying cells requires maintaining conditions similar to the cells’ natural conditions. All the cell culture related methods were performed under sterile conditions utilizing laminar flow hood. All experiments involving cells were performed when the cells were 70-80% confluent, which in most cell lines corresponds to the logarithmic phase of growth when the cells are not experiencing any stress.

Cell Lines

In our study, variations of three different cell lines were used: - HeLa, Tert-RPE, and Hap1. The cell lines, the variation used, their denotation, and description are as follows:

 HeLa-468: Green Fluorescence Protein (GFP) - tubulin transduced into HeLa cells which are cancerous epithelial cells from the cervix.

 HeLa-563: Flag-tagged wild-type GCN2 transduced into HeLa-468 cells along with Blasticidin resistance using lentivirus.

 HeLa-565: Flag-tagged siRNA6-resistant GCN2 transduced into HeLa-468 cells along with Blasticidin resistance using lentivirus.

 TERT_RPE: Telomerase immortalized Retinal Pigment Epithelium (RPE) cells.

 TERT_RPE_565: Flag-tagged siRNA6-resistant GCN2 transduced into TERT-RPE cells along with Blasticidin resistance using lentivirus.

 HAP1: It is near-haploid cell line except for chromosomes 8, derived from the male chronic myelogenous leukemia (CML) cell line KBM-7.

 HAP1-GCN2Δ: Hap1 cells whose GCN2 gene has been deleted.

 HAP1-GCN2Δ_563: HAP1_GCN2Δcells transduced with Flag-tagged wild-type GCN2 along with Blasticidin resistance using lentivirus.

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Cell culture

The general procedure and the physical conditions applied to all of these cells were the same except for the culture media used.

The specific media used for each of these cell lines are summarized below and will be referred to as growth media for respective cells from now on.

Table 2.1 Cell lines and the growth media used for their culture.

S.No Cell line Media used

1 HeLa RPMI 1640 (Roswell Park Memorial Institute Medium) w L-glutamine + phenol red

2 Tert-RPE DMEM /F12 (Dulbecco's Modified Eagle Medium) 3 HAP1 IMDM (Iscove’s Modified Dulbecco’s Medium)

Among these, RPMI 1640 is suitable for cultures in suspension and monolayer for various cell types including HeLa (42). RPMI contains a high concentration of vitamins, glutathione - reducing agent, and a bicarbonate buffer system which requires 5% CO2 environment to maintain physiological pH.

However, it does not contain proteins, lipids, or growth factors that need to be supplied in the form of 10% Fetal Bovine Serum (FBS). Added glutamine is an additional source of reduced carbon and an important precursor in the Tricarboxylic acid (TCA) cycle and also helps prevent ammonia toxicity.

Phenol red is used as a pH indicator in the media.

Similarly, DMEM /F-12 media consists of a high concentration of glucose, amino acids, and vitamins along with the F12 coagulase factor required for RPE cells. However, it is similar in other

specifications to RPMI in that it uses the bicarbonate buffer system, glutamine, and phenol red and requires FBS for growth factors [43].

IMDM is suitable for highly proliferating, high-density cell cultures with selenium and additional amino acids and vitamins but lacks iron compared to DMEM.

Cells were cultured in either a T-25 flask or T-75 flask with their respective growth media + 10% FBS for growth factors, lipids, and other components and 100 U/ml antibiotics, combination of Penicillin and Streptomycin (PS) to prevent risk of contamination. These cells were incubated at 37 ⁰C with 5%

CO2 in a humidified incubator. The cells were sub-cultured to prevent overgrowth twice every week and single culture was only used for a month after taking them out of storage in liquid nitrogen to prevent the effect by genetic drift.

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Sub-culture

The mammalian cells cultured were naturally adherent and needed to be detached from the flask for sub-culture. For this, trypsin was employed. Trypsin is a protease that can degrade cell adhesion molecules and extracellular components to free the cells from matrix. Before applying the trypsin, the media needs to be removed and cells washed with Phosphate Buffer Saline (PBS) to remove residual FBS which contains a1-antitrypsin.

Protocol

1. Remove media from the flask using suction pump being careful not to remove adherent cells

2. Wash cells using PBS, warmed to 37 ⁰C. 5 ml PBS for T-25 flask and 10 ml PBS for T-75 flask.

3. Add 0.25% Trypsin in EDTA to the flask, 350 µl for T-25 flask and 500 µl for T-75 flask.

Make sure that trypsin has covered the culture area. The volume used depends on the time of incubation and needs to be enough to not let the cells dry.

4. Incubate at 37 ⁰C for suitable an amount of time. 5 minutes for RPE cells and 2 minutes for HeLa and Hap1 cells.

5. Check the detachment of cells using inverted light microscope. Cells should be seen floating if detached and if not, incubate for extra 1 minute.

6. Add pre-warmed growth media with FBS and PS to the cells to inactivate the trypsin and collect cells with this media after repeated pipetting. Similar volume to PBS (step 2) is needed.

7. Seed required proportion of the cell suspension collected for sub-culture in new flask.

8. Add new growth media to make up the required volume (5ml for T-25 flask and 10 ml for T-75 flask) and incubate the culture at 37 ⁰C with 5% CO2 in a humidified incubator.

Freezing and thawing of cells

Mammalian cells cannot be continuously subcultured for use. This would be expensive and with continuous division, genetic drift can arise. Thus, cells need to be stored which can be done by freezing the cells in a process known as cryopreservation. In this process, cells are frozen slowly in a growth media containing 10% FBS and 10% dimethylsulfoxide (DMSO). DMSO helps prevent ice crystal formation during freezing process [44]. However, thawing and culturing the cells should be done as quickly as possible to dilute DMSO, as it is toxic to cells.

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Protocol:

Cryopreservation of cells

1. Trypsinize and collect the cells that are in logarithmic phase (70-80% confluent).

2. Determine the concentration of cells in the cell suspension using manual or automated cell counting.

3. Calculate total number of cells in suspension.

Total number of cells = Concentration of cells X volume of cell suspension in ml 4. Centrifuge the suspension at 2000 rpm for 5 minutes and remove media.

5. Add the new freezing media made of growth media + 10% FBS + 10% DMSO to the pellet of the cells and mix properly. Maintain over 2*10⁶ cells per ml.

6. After thoroughly mixing, aliquot 0.5 ml of the suspension in each freezing vials.

7. Place the vials in a slow freezing box with isopropanol and place it in -80 ⁰C.

8. After the cells have frozen, move the vials to a nitrogen tank.

Thawing cells

1. Collect the frozen cells from the freezer in dry ice.

2. Pipette the appropriate amount of growth media with FBS and PS into the flask.

3. Warm the cells as quickly as possible using water bath at 37 ⁰C.

4. Seed 300 µl of the cell suspension for the T-25 flask or 500 µl for T-75 flask.

5. Incubate the flasks at 37 ⁰C at 5% CO2 in a humidified incubator.

6. After 24 hours, remove media and wash the cells with pre-warmed PBS. This is followed by addition of new media of the same volume.

7. Incubate the cells again at 37 ⁰C at 5% CO2 in a humidified incubator.

2.2 Cell count

Many experiments require certain number of cells to be used. For this, cells needs to be counted. We have two methods to count cells:

1. Automated counting 2. Manual counting 2.2.1 Automated counting

Countess^TM II automated cell counter was used to determine the total number of cells and viability percentage. Viability is determined using Trypan Blue stain which is only taken in by dead cells. When

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a 1:1 mixture of trypan blue stain and cell suspension is applied on the Countess Cell counting

chamber slide and analyzed, the machine will count the cells based on the size-limitations provided and the color of the cells to determine the number and viability of the cells in the suspension.

Protocol

1. Trypsinize and collect cells that need to be counted.

2. Pipette 10 µl of 0.4% trypan blue stain onto a small piece of paraffin tape.

3. Mix the cell suspension well to prevent aggregation of the cells and pipette 10 µl of cell suspension.

4. Mix the cell suspension with trypan blue by pipetting it repeatedly.

5. Apply 10 µl of the mix to the Countess Cell counting chamber slide and let it rest for 30 seconds.

6. Insert the slide into the counter and use the program specified for the cell type.

7. When the counter focuses on the cells, press scan.

8. Note the concentration provided and the viability percentage of cells in the mixture.

2.2.2 Manual counting

The error in concentration of cells determined using automated counter can be high. The main reason for this could be because of sub-optimal mixing of the cell suspension as the machine only observes a single area. And if the cell aggregates are present, it would not be the best representation of the cell suspension. So, in some conditions, manual counting of cells might be needed to

determine the concentration of cells after observing multiple fields. In manual counting, slides like the Burker chamber or the Neubauer hemocytometer are used for counting of the cells. These slides have grids of a specific area itched into them (Fig. 6), which when combined with the specific height of loading area of the slide (0.1mm), gives specific volume. Under a light microscope, this can be viewed and cells can be counted within that area, therefore within that volume. As shown in figure 6, cells observed in Burker chamber with volume of 0.1 µl can be counted. Generally, 5 of those areas are counted and average calculated to determine the concentration.

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Figure 6 Grids in the Burker chamber. Large squares have 1 mm sides giving area of 1 mm² and volume of 0.1 mm³ or 0.1µl.

Protocol

1. Trypsinize and collect cells that need to be counted.

2. Mix the cells properly to prevent aggregates and load 15 µl of cell suspension into the chamber.

3. Observe the chamber under the microscope using 20X objective lens and count the cells present in determined area.

4. Calculate the concentration of cells using formula:

Concentration of cells per ml = Number of cells counted X 10000

2.3 Transient transfection

Transfection is the process of introducing nucleic acid into a eukaryotic cell through a non-viral method [45]. In our project, we used the lipid-based transfection agent Lipofectamine RNAiMAX Transfection Reagent. The positively charged head of the lipid interacts with the phosphate backbone of the nucleic acid and facilitates the nucleic acid condensation. The positive surface charge of the newly formed liposome mediates interaction between nucleic acid and cell membrane which is followed by endocytosis to take the nucleic acid inside the cell [46].

We transfected our cells with small interfering RNA (siRNA) which is a double-stranded RNA molecule with a 3’ overhang of 2 nucleotides. They bind to their target RNA and promote the degradation of the messenger RNA (mRNA) which decreases the amount of protein being translated [47]. During this experiment a negative control (siCTRL) is used to determine the effect of siRNA.

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Protocol

Day 1

1. Trypsinize, collect, and count the cells that are to be used for knockdown.

2. Seed 2*10⁵ cells in two 35 mm dishes in 2 ml of growth media+10% FBS+100U/ml PS. One for knockdown and another for control.

3. Incubate the cells 37 ⁰C at 5% CO2 in a humidified incubator.

Day 2

1. Mix 10 pmole of siRNA with 160 µl of reduced serum media - Opti-MEM, followed by 3 µl of Lipofectamine RNAiMAX reagent. Prepare mix for both siCTRL and siRNA.

2. Mix them properly by flicking and incubate at room temperature for 10 minutes.

3. In the meantime, prepare transfection media: growth media +10% FBS

4. Remove the old media from the culture dishes and add 1 ml of fresh transfection media.

5. After incubation of transfection mix, add 160 µl of mix into their respective dishes dropwise to cover the whole area.

4. Incubate the cells 37 ⁰C at 5% CO2 in a humidified incubator.

Day 3

1. Sub-culture the cells into two new dishes, one to be used for experiment and other for immunoblotting to test the knockdown efficiency.

2. Incubate the cells 37 ⁰C at 5% CO2 in a humidified incubator.

Table 2.2 The amount of each component to use for different culture dishes.

Culture dish

Number of cells seeded

Amount of siRNA

Volume of RNAiMAX reagent

Volume of OPTI- MEM

35 mm 2*10⁵ 10 picomole 3 µL 160 µL

60 mm 6*10⁵ 30 picomole 9 µL 500 µL

2.4 Investigation of Protein Expression

Immunoblotting is a widely used method of studying protein expression. The Figure 7 shows the general steps in immunoblotting.

16

Figure 7 Flowchart showing the steps in western blot. Modified from bosterbio.com.

2.4.1 Protein extraction

The first step in the analysis of the protein is its extraction from the cell. This is achieved by using appropriate lysis buffer to break the cell membrane and release the protein. The lysis buffer consists of different components suitable for the job. Detergent like TritonX-100 is responsible for breaking the cell membrane. Different salts like NaCl and MgCl2 are responsible for maintaining the

appropriate ionic strength of the solution which helps to stabilize the protein. Tris-HCl buffer prevents destabilization caused by pH change, as it has a buffer range of 7.0 to 9.0 pH. Lysis buffer also consists of EDTA which binds with metal ions thereby, preventing action of various proteases that uses metal ions as cofactors. This process is also helped by the presence of protease inhibitors [48]. Finally, the addition of benzonase - a nuclease, helps by degrading nucleic acids which

otherwise would result in viscosity of the final solution.

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Protocol

1. Remove media from the cell culture and place the culture dish in ice.

2. Add 80-100 µl of lysis buffer with benzonase into the dish.

3. Prepare cell scraper beforehand by washing it with water followed by ethanol and air dry.

4. Use the cell scraper to detach the cells from the dish surface.

5. Collect the cells with lysis buffer into an Eppendorf tube.

6. Incubate the lysate for 1 hour in ice or cold room.

7. Centrifuge the lysate solution at 13000 rpm for 10 minutes.

8. Transfer the supernatant into new Eppendorf tubes.

2.4.2 Protein measurement

Protein concentration in the lysate was measured using Bradford assay. It is based on the

colorimetric principle that Coomassie Blue G-250 when bound to proteins, changes color and the intensity of color change observed has a linear relation to concentration of protein present.

Therefore, it is possible to use Beer-Lambert’s law along with a standard curve to determine protein concentration in the sample. A standard curve can be generated using known concentration of Bovine Serum Albumin (BSA).

Protocol

1. To generate the standard curve and measure protein concentration, prepare mixtures in a 96 well plate as given in Table 2.3.

Table 2.3 Composition of each of the mixture for protein measurement.

Reaction mix Blank S1 S2 S3 S4 S5 Sample Lysis blank

BSA (2µg/µl) µl 0 1 2 3 4 8 - -

Sample solution (µl) - - - 1 1 (lysis buffer)

Milliq H2O (µl) 50 49 48 47 46 42 49 49

Bradford reagent 1X (µl) 100 100 100 100 100 100 100 100

Total volume (µl) 150 150 150 150 150 150 150 150

2. Incubate at 37 ⁰C for 30 minutes.

3. Measure the absorbance of all the mixtures at 595 nm using Thermoscientific multiscanFC.

4. Calculate the corrected absorbance for all the standard dilutions (S1-S5) by subtracting absorbance of the blank from their absorbance.

5. Plot a graph with absorbance against BSA amount used.

18

6. Calculate the equation for the line of best fit in the form of Y=aX + B where ‘X’ is the

amount of protein applied, ‘Y’ is the absorbance, ‘a’ is the slope of the line and ‘B’, is the y-intercept.

7. Calculate the corrected absorbance for the sample by subtracting absorbance measured for lysis buffer (Lysis blank) from that of the sample.

8. Use this corrected absorbance in the equation calculated from the standard curve to determine the protein concentration of the sample.

2.4.3 Protein separation

Different proteins have different physiochemical properties like size, net charge, or isoelectric point.

Using these properties, proteins can be separated from a mixture and analyzed. The most widely used method of separation is sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE).

Electrophoresis is the phenomenon of movement of charged particles towards the electrode

corresponding to the opposite charge under the influence of an electric field. Thus, the movement of the molecules being separated depends on the charge present. Polyacrylamide gel (PAG), on other hand, is a neutrally charged, hydrophilic matrix of polymer. Based on the amount of polyacrylamide used, the pores in the matrix can be regulated and used for separating proteins based on their size.

To remove the effect of protein structure and the charge present in the proteins, the sample is boiled in a sample buffer consisting of SDS and reducing agent dithiothreitol (DTT). SDS is an anionic

detergent which, when bound to protein, can break hydrogen bond and impart negative charge to proteins. DTT on the other hand, is capable of breaking strong disulfide bonds and together turning proteins into a linear structure with an evenly distributed charge per unit mass [49].

Protocol

1. Based on the calculated protein concentration, take the required volume (for 30 µg protein) of the protein extract and add lysis buffer to a volume of 21 µl

2. Add the required amount of 4X loading buffer with 1M DTT to get final 1X concentration.

4X loading buffer: 1M DTT = 5:1, volumes used are 7.5 µl:1.5 µl.

3. Boil the sample at 95 ⁰C for 5 minutes in a heating block.

4. Prepare set-up for SDS-PAGE using laemmli buffer as the running buffer at pH 8.0.

5. Load samples in the gel of choice (4-15% polyacrylamide precast gel).

6. Load 7 µl of Precision Plus Protein^TM Dual-color standard in one of the well.

7. Connect the electrical supply and run the gel at a constant voltage of 160 volts for 15 minutes and then at 200 volts until the 25 kDa marker reaches the end of gel.

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2.4.4 Protein transfer

Proteins separated by SDS-PAGE need to be transferred to a membrane, in our case a polyvinyl difluoride (PVDF) membrane, for immunodetection. Proteins cannot bind to PVDF membrane as it is.

PVDF need to be covered in ethanol to activate the positive charge in the membrane which then can bind with the negative charge of the protein. This binding is irreversible for the most part. For the transfer of the protein from the gel to the membrane, semi-dry transfer was used. In this process, a Filter-Gel-Membrane-Filter sandwich is prepared and the current conduction between the filters is responsible for transferring proteins from the gel to the membrane. Filters are soaked with a transfer buffer called Towbin buffer. It is a Tris-glycine buffer of pH 8.3 and consists of ethanol and SDS.

Ethanol helps to prevent swelling of the gel during transfer due to heat while SDS helps the protein to move out of the gel and to the PVDF membrane [49].

Protocol

1. Clean tray and tools before starting the work.

2. Take a PVDF membrane and soak it in ethanol for a minute.

3. Collect the ethanol and soak the membrane in water until the membrane is translucent.

4. Remove water and add transfer buffer to the membrane; leave it at room temperature until needed.

5. Take two pieces of filter and soak them in transfer buffer.

6. Take the gel after SDS-PAGE is complete and remove gel boarders as well as the gels between wells for the ease the handling.

7. Submerge the gel in the transfer buffer and incubate at room temperature for 10 minutes.

8. Assemble the sandwich of Filter-Gel-Membrane-Filter from top to bottom in the transfer cassette. Take extra care to remove any air pockets.

9. Remove extra liquid and close the cassette.

10. Place it in the Trans-Blot®Turbo™ Transfer system (BIO-RAD) and select the program to be run. In our case, the high molecular-weight (MW) for gel being used (midi or mini).

High MW program refers to 10 minutes run at 2.5A constant current with maximum of 25V.

2.4.5 Immunodetection

To analyze the transferred protein, it needs to be visualized. This was done by using enhanced chemiluminescence. In this method, proteins are probed with a protein-specific primary antibody.

Then these are once again probed with HorseRadish Peroxidase (HRP)-bound secondary antibodies

20

specific for the primary antibody. HRP is an enzyme that oxidizes luminol in presence of Hydrogen Peroxide (H2O2) giving a luminescent product emitting light. This light is detected by a camera and the image is then analyzed to assess protein abundance based on band intensity using softwares like Imagelab or ImageJ. To prevent unspecific binding, blocking agent (5% dry skim milk) is used before probing with primary antibody while the membrane is washed thoroughly after use of each antibody [49].

Protocol

1. Remove the membrane from the cassette and place it on a tray with Tris – buffered saline with Tween (TBS-T).

2. Remove TBS-T and apply the blocking solution (5% milk in TBS-T).

3. Incubate at room temperature for 1 hour.

4. Remove blocking solution and apply the primary antibody and incubate overnight at 4 °C in a tilting tray.

5. Collect primary antibody and wash the membrane 3 times with TBS-T, 10 minutes each.

6. Apply secondary antibody and incubate at room temperature for 1 hour.

7. Remove secondary antibody and wash the membrane three times with TBS-T, 10 minutes each.

8. Prepare substrate solution A:B in 1:1 (v/v)solution (Immobilon™ Western, Chemiluminescent HRP substrate).

9. Add substrate to the membrane and wait for 5 minutes.

10. Visualize the signal using ChemiDoc^TM MP Imaging system for chemiluminescence and visual light.

11. Quantify the band intensity using Imagelab.

2.5 Analysis of gene expression

One of the methods used in the analysis of transcript levels is quantitative Polymerase chain reaction (qPCR). Figure 8 shows the workflow of the qPCR. The process starts with the collection of cells followed by RNA isolation. Isolated RNA is used for reverse transcription to form complementary DNA (cDNA). This cDNA is then amplified using polymerase chain reaction (PCR) while measuring the amount of DNA formed in each cycle. This result is then analyzed.

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Figure 8 Workflow of qPCR analysis. (Copyright Ståhlberg et al., 2011)

2.5.1 RNA isolation from cells

The very first step in the analysis of RNA levels is RNA isolation. For this MasterPure™ Complete DNA and RNA Purification Kit was used. The kit is a chemical-based isolation method that uses different enzymes to breakdown components of the cell and the solubility difference between molecules to separate them. Based on the recommendation provided, the maximum number of cells that could be used for RNA isolation (10⁶) was counted and collected. These cells were lyzed using the tissue and cell lysis buffer provided in the kit along with Proteinase K, which degrades proteins. Proteins were then precipitated using the precipitation solution and centrifugation. Isopropanol is used to

precipitate nucleic acids from the supernatant. DNA present in the supernatant was degraded using DNase I, a nuclease and what remains was isolated total RNA. All the steps in this method need to be done with proper care, starting with a clean bench and use of gloves in every step to prevent any RNases from contaminating the sample.

Protocol

A. Cell collection

1. Trypsinize and collect the cells.

2. Count the cells using Countess^TM II automated cell counter.

3. Based on the concentration, make aliquots each containing 10⁶ cells in Eppendorf tubes.

4. Centrifuge the cells at 200 rpm for 5 minutes at 4 °C.

5. Remove all the media and put the Eppendorf tubes directly in liquid nitrogen for storage.

B. RNA isolation

1. Dilute 1 µL of Proteinase K, into 300 µL of Tissue and Cell Lysis Solution for each sample.

22

2. Add 300 µL of Tissue and Cell Lysis Solution containing the Proteinase K and mix

thoroughly.

3. Incubate at 65 °C for 15 minutes; vortex every 5 minutes.

4. Place the samples on ice for 3-5 minutes.

5. Add 150 µL of MPC Protein Precipitation Reagent to 300 µL of lysed sample and vortex vigorously for 10 seconds.

6. Pellet the debris by centrifugation at 4 °C for 10 minutes at 13000 rpm in a microcentrifuge. If the resultant pellet is clear, small, or loose, add 25 µL of MPC Protein Precipitation Reagent, mix, and pellet the debris again.

7. Transfer the supernatant to a clean microcentrifuge tube and discard the pellet.

8. Add 500 µL of isopropanol to the recovered supernatant. Invert the tube 30-40 times.

9. Pellet the total nucleic acids by centrifugation at 4 °C for 20 minutes in a microcentrifuge.

10. Remove all the residual isopropanol with a pipette.

11. Prepare 200 µL of DNase I solution for each sample by diluting 5 µL of RNase-Free DNase I up to 200 µL with 1X DNase Buffer.

12. Completely resuspend the total nucleic acid pellet in 200 µL of DNase I solution.

13. Incubate at 37°C for 10 minutes. Note: Additional incubation (up to 30 minutes) may be necessary to remove all contaminating DNA. 5. Add 200 µL of 2X Tissue and Cell Lysis Solution; vortex for 5 seconds.

14. Add 200 µL of MPC Protein Precipitation Reagent; vortex for 10 seconds; place on ice for 3-5 minutes.

15. Pellet the debris by centrifugation at 4 °C for 10 minutes at 13000 rpm in a microcentrifuge.

16. Transfer the supernatant containing the RNA into a clean microcentrifuge tube and discard the pellet.

17. Add 500 µL of isopropanol to the supernatant. Invert the tube 30-40 times.

18. Pellet the purified RNA by centrifugation at 4°C for 20 minutes in a microcentrifuge.

19. Carefully pour off the isopropanol without dislodging the RNA pellet.

20. Rinse twice with 70% ethanol, being careful to not dislodge the pellet. Centrifuge briefly if the pellet is dislodged. Remove all the residual ethanol with a pipette.

21. Resuspend the RNA in 10-35 µL of TE Buffer.

22. Add 1 µL of RiboGuard™ RNase Inhibitor.

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2.5.2 RNA measurement

After isolation of the RNA, it is checked for both quality and quantity before further experimentation.

The quality check was performed using the NanoDrop ND-1000 Spectrophotometer (Nanodrop Technologies). This is a spectrophotometer which measures the concentration of the sample based on their absorbance characteristics. To measure the concentration of the nucleic acids, the device measure absorbance at 230 nm, 260 nm, and 280 nm. Measurements made at 260 nm are used to determine the concentration of nucleic acid present in the sample. The concentration of RNA is calculated as 40*Absorbance at 260nm. As the aromatic amino acids are mainly responsible for the absorbance at 280 nm, measurements made at this wavelength give the concentration of protein present and the ratio of absorbance at 260:280 nm provides the rough estimation on protein contamination in a nucleic acid sample. A ratio of around 2.0 is a nucleic acid sample with very little protein contamination. Similarly, absorbance at the wavelength 230 nm is the result of the presence of salt in the sample and a ratio of the absorbance at 260nm to 230 nm gives estimation for the salt contaminants in the sample. Acceptable values for this ratio are also around 2.0.

Protocol

1. Start the program and choose “measurement of nucleic acid¨.

2. Load 2 µl of RNAse-free water into the lower measurement pedestal while keeping the sample arm open.

3. Close the arm and press ok in the program. This lets you choose between RNA and DNA measurement.

4. Wipe the lower measurement pedestal with filter paper and load TE buffer.

5. Close the sample arm and calibrate this as a blank.

6. Follow the steps 4 and 5 for each sample but just measure the values and make a report of both quality and quantity of the RNA.

7. Before closing, perform the spectroscopic measurement of the nuclease-free water and wipe the pedestals with filter paper.

2.5.3 Reverse transcription

After quantifying the RNA, it is used for reverse transcription to synthesize cDNA which will be used for qPCR. This process converts all mRNA to cDNA while maintaining the differences between the levels of the mRNA for all the genes. We used the High-Capacity cDNA Reverse Transcription Kit (applied biosystems) to perform reverse transcription. This kit is capable of reverse transcribing up to 2 µg of RNA into single-stranded cDNA. This kit utilizes a master mix with a MultiScribe™ Reverse Transcriptase that transcribes a template of mRNA into cDNA using random primers, which are

24

oligonucleotides with random sequences. These primers are necessary for binding of reverse

transcriptase to the mRNA and are not limited by the requirement of polyA tail. After binding, the enzyme will start adding nucleotides complementary to the mRNA. After the completion of the reverse transcription, the mRNA and the cDNA strand formed are separated at a temperature of 85

°C. Volume equivalent to 2 µg of RNA was taken and diluted to 10 µl using nuclease-free water. The reagent solution was prepared according to the manufacturer’s instructions and added to the RNA sample. The PCR tubes were then loaded in a thermocycler with recommended conditions and run.

The recommended composition of the solution and conditions for the thermocycler are given in Table 2.4 and 2.5.

Table 2.4 Composition of solutions for reverse transcription.

Master mix Initial concentration Final concentration Volume added

RT Buffer 2X 1X 2.0 µL

dNTP Mix (100 mM) 100 mM 4 mM 0.8 µL

RT random primers 10X 1X 2.0 µL

MultiScribe™ Reverse transcriptase

50 U/µL 5.0 U/µL 1.0 µL

RNase Inhibitor 20 U/µL 1.0 U/µL 1.0 µL

Nuclease free water 3.2 µL

Total* 10 uL

*Add 10 µL of RNA solution to 10 µL of the master mix. Maximum 2 µg of absolute RNA

Table 2.5 Setting in the thermocycler for reverse transcription.

Cycling conditions Step 1 Step 2 Step 3 Step 4

Temperature (°C) 25 37 85 4

Time (minutes) 10 120 5 ∞

2.5.4 Testing the product of reverse transcription

To test the success of reverse transcription, the product was used as a template for a polymerase chain reaction (PCR). PCR is a method that is used to amplify the DNA of interest using a specific pair of primers. We also use PCR to test the ability of primers to form a single product while testing the suitability of all the primers that were used. PCR was followed by capillary electrophoresis to detect the product of PCR. PCR works in a three-step process.

1. Denaturation: Double-stranded DNA is separated into single-stranded DNA 2. Annealing: Primers and the DNA polymerase enzyme bind to DNA.

25

3. Elongation: DNA polymerase enzyme adds deoxynucleotides (dNTPs) in the 3’-end of the

primer, complementary to the template DNA being used.

These three steps are repeated a number of times, usually 30-40, and with each replication the amount of the target DNA doubles. This is a basic method to amplify DNA of interest from a limited sample. In this experiment, we used Platinum™ II Taq Hot-Start DNA Polymerase kit for PCR reaction.

This kit consists of a DNA polymerase that is an engineered Taq DNA polymerase with an increased resistance to reaction inhibitors originating from sample material or DNA purification steps. The polymerase mix also contains MgCl2 equivalent to make it 1.5mM in the final reaction [50]. The reaction mix was prepared and loaded in the thermocycler with reaction settings based on recommendation from the kit. The recommendations are given in Table 2.6 and 2.7.

Table 2.6 Composition of the reaction mix for PCR.

Master mix Initial concentration Final concentration Volume added

Platinum™ II PCR Buffer 5X 1X 4 µL

Forward primer 10 µM 0.2 µM 0.4 µL

Reverse primer 10 µM 0.2 µM 0.4 µL

dNTP Mix 10 mM 0.2 mM 0.4 µL

Platinum™ II Taq Hot-Start DNA Polymerase

5 U/µL 0.04 U/µL 0.16 µL

Template cDNA <500ng 1 µL

Nuclease free water 13.64 µL

Total 20 µL

Table 2.7 Settings in the thermocycler for PCR.

Step 1:

Initial Denaturation

Step 2:

Denaturation

Step 3:

Annealing

Step 4:

Extension

Step 5:

Final Extension

Temperature (°C) 94 94 60 68 68

Time (minutes) 2 0.25 0.25 0.25/kb 10

Cycles 1 30 1

The product of a PCR reaction can then be checked using electrophoresis to separate DNA based on size and to visualize it. In our project, QIAxcel Advanced System, which is a rapid analysis system that uses capillary electrophoresis to separate samples based on sizes was used. The instrument includes an array of light-emitting diodes and micro-optical collectors that latch to capillaries within QIAxcel gel cartridges. Fragments of the sample being analyzed move through a gel matrix within the capillary. This passage excites and produces detection spots causing a signal generation which is transmitted through a photomultiplier tube to the QIAxcel ScreenGel software for data

26

interpretation [51]. Use of reference markers, 15 bp and 5000 bp can be used to determine the size of DNA fragments present in the sample.

Protocol

1. Load the gel cartridge.

2. Fill and load the buffer tray with the QX Separation Buffer.

3. Load the samples in PCR tubes, a minimum of 3 µl of sample is needed.

4. Load the reference marker in the reference tray.

5. Select the process profile to be used and go.

2.5.5 Real-time quantitative PCR

After making sure that the reverse transcription has worked, cDNA can then be used in further analyses using qPCR. The qPCR, also known as real-time (RT) PCR because it follows same principle as conventional PCR with only difference being the amount of DNA formed is tracked after reaction cycle. This is possible using specific fluorescent dye like SYBR®Green, which can be excited using illumination at a 494 nm wavelength of light and when bound to the DNA has a dampened

intramolecular motion, resulting in production of fluorescence of 521 nm to release the extra energy.

This fluorescence is detected by the fluorescence detector and kept track of throughout the reaction.

SYBR®Green in free form does not produce fluorescence and the produced fluorescence has linear relationship with the amount of double-stranded DNA formed [52].

Quantification of a sample using qPCR can be done in two different ways: absolute quantification and Rrlative quantification. We used the relative quantification method to measure the cDNA present in the sample and refer this value to the transcript level. In this method, serial dilution of an undiluted sample is formed which is then used to make a standard curve for a particular primer set. Then this standard curve can be used to find the relative amount of that particular cDNA. However, to properly understand and compare the observed values, these need to be normalized to a reference gene, also called housekeeping gene [53]. In our case, we used TATA-Binding Protein (TBP) and Glucoronidase beta (GUSB) as housekeeping genes.

We used the Applied Biosystems StepOne^TM Real-Time PCR system, both instrument and software, along with SYBR®Green PCR Master Mix (Applied Biosystems) for quantification of cDNA. This system uses OneStep software to calculate the quantity of DNA present in the sample. It does so by

calculating the Threshold cycle (CT)value, which is the number of cycles in an amplification at which the fluorescence in the sample is above the background level. This value, along with the efficiency of the primer is used by the software to calculate the quantity of the DNA present in the sample.

27

Efficiency of a primer pair is their ability to produce two new DNA molecules from one double-

stranded DNA molecule in every cycle. For a primer pair to be used in a qPCR experiment, they need to have efficiency in the range of 90-110% [54].

Another aspect to consider while recording the qPCR result is the irregularities in the amplification curve provided by the software for each sample. In this curve, normalized fluorescence signal is plotted against the number of cycles. We need to ascertain that the returned value of the sample falls within measured points of the standard curve. Any points outside this range cannot be used reliably. We also have to look into the regression coefficient of the standard curve and if it is not close to 1, then those standard curves cannot be used. Finally, the melt curve is inspected, which provides information on the specificity of the primer pair used. Multiple peaks in this curve represent an unspecific binding and such primer pair should not be used.

To perform the experiment, the solutions as given in Table 2.8 were made based on the recommendation from the manufacturer.

Table 2.8 Composition of reaction mix for qPCR.

Master mix for single pair of primer Volume used (µL) Final concentration

Power SYBR®Green Master Mix (2X) 7.5 1X

Primer pair mix (10µM) each 0.15 100 nM

Template* 1 -

MiliQ water 6.35 -

Total 15

* Template is added later in the reaction mix.

Protocol

1. Prepare the required number of serial dilutions from undiluted sample.

2. Using a Multipipette® M4, pipette out 14 µL of reaction mix in the required number of wells in a MicroAmp™ Fast Optical 96-well Reaction Plate.

3. Load the dilutions prepared separately for the standard curve and all the samples with technical duplicates.

4. Add 1 µL of sample in respective wells.

5. Cover the plate with a MicroAmp™ Optical Adhesive film.

6. Centrifuge the plate briefly to allow the mix to reach the bottom of the well.

7. Load the plate on the instrument and run the program for Power SYBR®Green Master Mix with standard ramp speed of about 2 hours and run-setup as givn in table 2.9.

28

Table 2.9 Setting in the OneStep device for qPCR.

Step 1:

Initial denaturation

Step 2:

Denaturation

Step 3:

Annealing

Step 4:

Melt curve (step and hold)

Temperature (°C) 95 95 60 60 95

Time (minutes) 10 0.25 1.00 0.3°C /15 seconds

Cycles 1 40 1

8. After completion of the reaction, export the results in an excel file.

9. Normalize the values of genes of interest to the values of the respective housekeeping gene.

10. Normalize the value obtained to the control cell line to observe the difference.

2.6 Functionality of the transduced GCN2

Methods employed during assessing the functionality of the transduced GCN2 (results 4.3.).

 UV irradiation of the cells

 Cell lysis and collection

 Immunoprecipitation of protein of interest and its substrate separately

 In vitro kinase assay

 Immunoblotting to observe the phosphorylation of the substrate 2.6.1.1 UV irradiation of the cells

In this work we irradiated cells with UV-radiation in order to activate GCN2, since it has a

constitutively low activity in unstressed cells. To determine the amount of time the cells need to be exposed to UV-radiation to deliver a given dose of radiation, a radiometer is used. We also must remember that the source of the UV-radiation in the safety cabinet does not emit UV-radiation uniformly to all the areas, cells must be irradiated in the same area where the radiometer was placed.

Protocol

1. Remove medium from cell culture dish and irradiate one of the samples with 60 J/m² UV- radiation.

Calculate the UV irradiation as follow:

2. Measure the UV-radiation in the hood using a radiometer. The readings are in mW/cm². 3. Calculate the amount of time required for exposing cells to the necessary 60 J/m² of UV

radiation by using formula: x=6000/M, where M is the measurement made in mW/cm² and X stands for time required in seconds for the required amount of radiation.

29

4. Add back the removed media to the culture dishes and incubate for 30 minutes at 37 ºC.

2.6.1.2 Cell lysis and collection

Similar to any other protein methods, cells need to be lysed for the in vitro kinase assay as well. The lysis buffer we used for this experiment is different than the general use lysis buffer because we want to test the protein activity and wants the protein to be stable in its native structure. We also want to preserve the phosphorylation state of the protein as it is one of the regulators of protein activity [55].

The composition of this lysis buffer and the function of each of the component are in TABLE 2.10.

Table 2.10 Different components in lysis buffer and their function.

Component Function

Calyculin A A marine toxin that inhibits phosphatases enzymes Protease inhibitors Inhibits proteases

HEPES* Buffer system; good for maintaining protein stability in low temperature Glycerol Increases protein solubility and stability

EDTA Reduce oxidation damage and chelate metal ions Triton X-100 Detergent that breaks down the cell membrane NaCl Salt to regulate osmolarity

*HEPES: 4-(2-hydroxyethyl)-1-piperazineethanesulfonic acid Protocol

1. Add 0.7 ml lysis buffer with calyculin A into all the three cultures (Control cell-line, UV irradiated cell line and PP1γ-GFP (substrate) cell line) and scrape the cells using a cell scraper.

2. Collect the cells after mixing by pipetting up and down into an Eppendorf tube.

3. Incubate at 4 °C for 20 min (rotating) 4. Centrifuge at 13000 rpm 4 °C for 10 min.

5. Transfer supernatants to new tubes: label as soluble extracts.

6. Save 25 µl of the soluble extracts in new tubes, add 25 µl 2X sample buffer: label as input.

2.6.1.3 Immunoprecipitation

Immunoprecipitation is a method in which an antibody specific for the protein of interest is used to precipitate that particular protein. It can be achieved by binding an antibody to a magnetic bead which is then mixed with a protein extract. During incubation, the antibody and the protein will make a complex which can then be isolated using a magnet. These reactions must be performed using a suitable buffer so as not to destabilize the protein. In our project, our proteins of interest, GCN2 and