Additional file 1 of Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas

Additional file 1. Supplemental methods: combinatorial drug screen and stable isotope tracing analysis of 13C6-glucose and 13C5-glutamine

Additional file 2 of Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas

Additional file 2: Figure S1. (A-B) A375R1 cells were seeded in 96 well plates (103 cells/well) and treated with GSK (A) or BKM120 (B) and their combinations with IACS at 1:1 concentration shown on the x-axes. Cell growth inhibition was determined after 72 h using Cell Titer Blue reagent. (C) The same experiment as above was performed on normal epidermal melanocytes with the indicated treatments, and cell growth inhibition was determined after 72 h using Cell Titer Blue reagent. (D) The same experiment as above was performed in A375R1 cells with the indicated inhibitors, but in this case, cell growth inhibition was determined after 72 h using Crystal Violet dye staining. In panels A-D, data is normalized to vehicle-treated cells and is average of triplicates, with error bars representing SD, and colored asterisks representing significant differences (*=<0.033; **=<0.002; ***=<0.001) in effects for combination treatment versus individual probes (red) or IACS (black). (E and F) A375R1 and UCSD354L cells were treated with 100 nM Dabrafenib (DAB) or its combination with IACS (100 nM) or STN (1 μM) for 72 h and cell cycle profiles were generated using propidium iodide-FACS analysis, which included sub-G1 (dead cell) population. Data are plotted as bar graphs of triplicates; error bars represent SD; Asterisk (*) represents significant differences (p<0.05) of DAB+STN compared to the other treatments shown. (G-J) Western blot bands from P-AKT_Thr308 and P-AMPK_Thr172 protein staining were quantified using Image J software and represented as bars graphs of quantified area in square pixels for each of the protein bands (y-axis) versus treatments (x-axis) for A375R1 (G and H) and UCSD354L (I and J) cells. Figure S2. (A, B) The Seahorse fuel-flex assay was performed on MEL624 (A) and WM1799 (B) cells to determine their dependency (blue) on glucose (GLC), glutamine (GLN) and fatty acids (FA), and their flexibility (orange) to utilize either of the single nutrients when the other two are inhibited. Data is quadruplicates; error bars, SD. (C, D) MEL624 (C) and WM1799 (D) cells were seeded in 96 well plates (103 cells/well) and treated with IACS or STN or their combinations at 1:1 concentration shown on the x-axes. Cell growth inhibition was determined after 72 h using Cell Titer blue reagent. Data is normalized to vehicle-treated cells and is average of triplicates, with error bars representing SD. (E) A375R1 cells were seeded in 96 well plates (103 cells/well) and treated with ETMR or STN at the concentrations shown on the x-axes. Cell growth inhibition was determined after 72 h using Cell Titer blue reagent. Data is normalized to vehicle-treated cells and is average of triplicates, with error bars representing SD. (F) A375R1 cells (1.5x104) seeded in 96 well plates were subjected to the same consecutive inhibitor treatments and incubation conditions as in Fig. 4D. The cells were then detached by trypsinization, live cells were counted using trypan blue dye exclusion and the results plotted as bar graphs of viable cells following each subsequent treatment. Data is triplicates; error bars, SD. (G) Relative abundance of intracellular acetyl CoA (Ac-CoA) in A375R1 cells treated with vehicle (V), IACS (I), STN (S) or their combination (I+S) for 12 hours. (H, I) Fractional labeling of acetoacetate by [U-13C]-GLC (glucose) (H) or [U-13C]-GLN (glutamine) (I) following treatment with IACS (I) and STN (S) or their combination (I+S) for 12 h. Figure S3. (A) Heatmap showing unsupervised clustering analysis of significantly (p<0.005) altered proteins in UCSD354L tumors harvested from mice fed with regular (Reg) or high-fat keto diet and analyzed by RPPA. The heatmap shows Pearson correlation of significantly (p<0.005) different proteins in tumors from mice fed with high fat keto (Hi-fat) versus regular (Reg) diet. Intensity ranges of lowest (blue) and highest (red) protein levels are indicated at the bottom of the heatmap. (B) Sub-cutaneous tumor growth of parental A375 cells in mice fed with Regular diet or high-fat keto diet. (C and D) Percent changes in body weights of A375R1 tumor bearing mice fed with regular (Reg) diet (C) or high-fat keto diet (Hi-fat) (D), and treated with Vehicle, 5mg/kg IACS, 1mg/kg STN or their combinations over the number of days shown. Mice weight data is from eight mice/group, error bars, standard error of mean (SEM)

Additional file 3 of Combined inhibition of HMGCoA reductase and mitochondrial complex I induces tumor regression of BRAF inhibitor-resistant melanomas

Additional file 3: Table S1. Inhibitors (probes) in the combinatorial drug screen, their molecular targets and cellular pathways targeted. Table S2. IC50 values of single agent inhibitors (probes) and their combinations with IACS-010759 (IACS), derived from growth inhibition curve analysis using GraphPad Prism

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Supplementary Methods from The Glutaminase Inhibitor CB-839 (Telaglenastat) Enhances the Antimelanoma Activity of T-Cell–Mediated Immunotherapies

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Supplementary Figures from A Novel Mitochondrial Inhibitor Blocks MAPK Pathway and Overcomes MAPK Inhibitor Resistance in Melanoma

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Supplementary Tables from A Novel Mitochondrial Inhibitor Blocks MAPK Pathway and Overcomes MAPK Inhibitor Resistance in Melanoma

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