6. Conclusion
6.1 Future perspective
Some of the results in this thesis are basic research, making room for many interesting experiments for future investigation.
We speculate whether MITF is a direct target for tankyrase inhibition or if MITF and tankyrase are connected. To better understand how the G007-LK mechanism affects MITF and tankyrase protein level, it would be interesting to use immunoprecipitation (IP) to isolate MITF and
tankyrase antigen from a mixture, followed by immunoblot analysis for detection. It could also be helpful to utilize mass spectrometry (MS) analysis to assess whether MITF and tankyrase are connected.
Knockdown and over-expression experiments of MITF siRNA and treatment with tankyrase inhibitor and WNT activating ligands could be interesting to explore if this affects YAP expression and vice versa. Furthermore, we are curious to investigate the effect of G007-LK, anti-PD-1, and combinational therapy with G007-LK + anti-PD-1 in mice parallel with MITF and YAP knockdown and overexpression experiments. A theory might be that overexpression of MITF leads to increased sensitivity towards anti-PD-1 or that a knockdown of MITF decreases the synergic effect of G007-LK and anti-PD-1 demonstrated in previous research [25].
63 Our experiments established that MITF is not linked to β-catenin when regulated, suggesting another mechanism involved. Immunofluorescent staining did not indicate a co-localization of either MITF/β-catenin protein or MITF/YAP protein. To further explore the underlying regulating mechanism of MITF upon treatment with G007-LK, it can be useful to investigate several more antibodies, such as anti-TNKS1/2 and anti-AMOTL1/2, for their determination of localization together with MITF. It is still unknown where MITF is located in the WNT and YAP signaling context.
It would be interesting to investigate more antibodies for immunoblotting of melanoma, for example, PGC-1α, which is a co-activator for several different transcription factors – including MITF via α-MSH [124]. Previous research has shown that overexpression of PGC-1α in B16-F10 melanoma cells led to hyperpigmentation of the cells [124]. PGC-1α regulates the Tyrosinase gene directly, which plays an essential role in melanocyte development [80] [124]. MITF
controls tyrosinase, and a PGC-1α knockdown has reduced MITF-M expression, and MITF protein in B16-F10 and several human melanoma cell lines [124].
Additionally, it would be interesting to explore the effect of G007-LK on MITF target genes for differentiation, proliferation, and survival. More than 40 MITF target genes have been identified, listed in Appendix C, Supplementary table 9 [125].
64
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Appendix A: Recipes
Supplementary Table 1. SDS loading buffer.
Supplementary Table 2. 10x Protein transfer buffer.
Supplementary Table 3. 1x Protein transfer buffer.
Supplementary Table 4. 4% PFA (1L) [126].
1L 4% PFA
Reagent Volume
1X PBS (pH 7.4) 800 mL Paraformaldehyde powder 40 g
NaOH 1 N
Dilute HCl X
SDS loading buffer
Reagent Volume
1M Tris-HCl (pH 6.8) 2 mL
SDS 0.8 g
14.7 M β-mercaptoethanol 4 mL
0.5 EDTA 1 mL
Bromophenol Blue 8 mg
H2O 2 mL
10x Protein transfer buffer
Reagent Volume
Trisma-base 30.3 g
Glysin 144 g
dH2O 1000 mL
1x Protein transfer buffer
Reagent Volume
10x Protein transfer buffer 100 mL
Methanol 200 mL
dH2O 700 mL
72 Supplementary Table 5. Primary antibodies diluted in 4% BSA/PBS.
Supplementary Table 6. Aqueous Mounting Medium [127].
Appendix B: Equipment and instruments
Supplementary Table 7. List of equipment.
Product name Catalog number Provider Country
Cell culture
B16-F10 ATCC® CRL-6475™ American Type Culture
Collection
FBS 10270-106 Life technologies Carlsbad, California, USA
P/S P4333 Sigma-Aldrich Saint Louis, Missouri, USA
PBS Oslo University hospital Oslo, Norway
Trypsin T3924 Sigma-Aldrich. Saint Louis, Missouri, USA
CryoTube 368632 Thermo Fisher Scientific Waltham, Massachusetts,
USA
Mr. Frosty 5100-0001 Thermo Fisher Scientific Waltham, Massachusetts,
USA
Mycoplasma detection kit LT07-318 Lonza Bazel, Switzerland
Poly-L-Lysine solution Sc-286689 Santa Cruz Dallas, Texas, USA
96 well plate 734-2073 NuncTM Roskilde, Denmark
6 well plate 140675 NuncTM Roskilde, Denmark
20 cm dishes NuncTM Roskilde, Denmark
T25 culture flask 156367 NuncTM Roskilde, Denmark
T75 culture flask 10364131 Thermo Fisher Scientific Waltham, Massachusetts,
USA Primary antibodies diluted in 4% BSA/PBS
Antibody Dilution rate
β-Catenin 1:200
MITF 1:50
YAP 1:200
Aqueous Mounting Medium
Kisser’s Kaiser’s Glycerol Jelly Modified
Gelatine 10 g 40 g 65 g 5 g
Distilled water 35 ml 210 ml 300 ml 50 ml Glycerol (glycerin) 30 ml 250 ml 100 ml 50 ml Phenol (carbolic acid) 5 ml 5 ml
73
T175 culture flask 10246131 Thermo Fisher Scientific Waltham, Massachusetts, USA
Eppendorf tubes 72.706 Sarstedt Nümbrecht, Germany
Cell scrapers 83.3959 Sarstedt Nümbrecht, Germany
Drugs and substanses G007-LK
DMSO D8418 Sigma-Aldrich Saint Louis, Missouri, USA
WNT3a CHIR
RT-qPCR
Qiagen RNeasy mini kit 74106 Qiagen Hilden, Germany
High-Capacity cDNA Reverse Transcription Kit
4368814 Applied BiosystemsTM Foster City, California,
USA
Master Mix 4370074 Thermo Fischer Scientific Waltham, Massachusetts,
USA
384 well plate 4343814 Thermo Fischer Scientific Waltham, Massachusetts,
USA Gapdh, TaqMan® Gene
Expression Assay, Hs02758991_g1
4131182 Thermo Fischer Scientific Waltham, Massachusetts, USA
Axin2, TaqMan® Gene Expression Assay, Hs00610344_m1
4331182 Thermo Fisher Scientific Waltham, Massachusetts, USA
Ctgf, TaqMan® Gene Expression Assay, Hs00170014_m1
4331182 Thermo Fisher Scientific Waltham, Massachusetts, USA
Cyr61, TaqMan® Gene Expression Assay, Hs00155479_m1
4331182 Thermo Fisher Scientific Waltham, Massachusetts, USA
4331182 Thermo Fisher Scientific Waltham, Massachusetts, USA
Immunoblot
NP40 lysis buffer FNN0021 Life technologies Carlsbad, California, USA
Protease inhibitor 4693116001 Roche Basel, Switzerland
RIPA buffer 89900 Thermo Fisher Scientific Waltham, Massachusetts,
USA
Non-fat dried milk A0830 Applichem Chicago, Illinois, USA
Non-fat dried milk A0830 Applichem Chicago, Illinois, USA