Fig 3. OXPHOS^high BAP1 mutant phenotype is linked to increased glycolytic-nucleotide biosynthetic pathway.

a. Protein expression of BAP1 and metabolic enzymes involved in glucose utilization and nucleotide synthesis in MM66, MP65 and MP38 cells. b. MM66, MP65 and MP38 cells were treated with WZB117 (12.5 or 25 μM) or 6-aminonicotinamide (6-AN, 31.25 or 62.5 μM) for 3 days. Cell viability changes were determined by crystal violet staining. Quantification of cell growth with WZB117 and 6-AN treatments in MM66, MP65 and MP38 cells presented as fold change in crystal violet staining of colonies. Representative crystal violet images of cell growth are shown. Scale bar: 100 μm. c. Re-expression of BAP1 in UM mutant cell clines, MP65 and MP46, was confirmed by western blot. MM66 serves as a positive control for BAP1 expression. d. The effect of BAP1 re-expression on the glycolytic capacity of MP65 cells was measured by ECAR using the Seahorse XF24 analyzer. Seahorse data were normalized to protein concentration and analyzed by Agilent Seahorse XF report generators. Data are shown as mean ± SEM (n=12). e. Metabolite percentage distributions within specific metabolic pathways that are increased or decreased in MP65 compared to MP65-BAP1 cells (FC >2, adj. p-val. < 0.05). f. Protein expression of metabolic enzymes involved in glucose utilization and nucleotide synthesis in MP65 and MP65-BAP1 cells. g. MP65 and MP65-BAP1 cells were treated with WZB117 (12.5, 25 or 50 μM) or 6-aminonicotinamide (6-AN, 31.25, 62.5 or 125 μM) for 5 days. Cell viability changes were determined by crystal violet staining. Quantification of cell growth with WZB117 and 6-AN treatments in MP65 and MP65-BAP1 cells presented as fold changes in crystal violet stain. Data are shown as mean ± SEM from biological replicate experiments (n=4). *p<0.05 and **p<0.01 unpaired t-test. ECAR; extracellular acidification rate, GLUT; glucose transporter, HK; hexokinase, G6PD; glucose-6-phosphate dehydrogenase and TKT; transketolase.