Supplemental Table 1: List of Cell Lines Used with Mutation Status and Vemurafenib Resistance

Supplemental Figure 1: Vemurafenib does not induce apoptosis in the majority of sensitive cells and does not significantly inhibit growth in acquired resistant subclones. A, 72 hour vemurafenib treatment does not induce significant amounts of apoptosis in sensitive lines. Sensitive YUMACs cells were incubated in either DMSO vehicle or 3μM vemurafenib for 72 hours and stained with conjugated Annexin V and propidium iodide to assess cell death. The percentage of cells that were not negative for both stains decreased by only 13.1% with drug treatment. B, Vemurafenib induces G0/G1 arrest in sensitive cell lines by 72 hours. Cells were treated with vehicle or 3μM vemurafenib before one hour BRDU pulse and four hour chase before fixation and analysis by flow cytometry. C, Vemurafenib did not greatly alter growth in acquired resistant subclones of two parental sensitive lines after 72 hours of incubation at 3μM. D, 3μM vemurafenib significantly decreased Erk activation in drug sensitive but not drug-resistant or non-BRAF mutant lines. *, p < 0.05; ***, p < 0.001.

Supplemental Table 1: List of cell lines used with mutation status and vemurafenib resistance.

Supplemental Figure 2: No significant change was observed in HKII mRNA in vitro with vemurafenib treatment or in immunohistochemical staining of patient biopsies. A, Cells treated with 72h 3μM vemurafenib showed less than 2-fold decrease in HKII mRNA levels. B, PRE vs. EDT patient biopsies showed minimal changes in HKII staining intensity. IHC data represents averaged scores for 100 cells per region with 5 regions scored per biopsy slide.

Supplemental Figure 3: Decreases in adherent cell area were observed in vemurafenib-sensitive cells, mirroring measurements of cell volume obtained by Coulter Counter. Cell area was calculated using ImageJ from captured 100X brightfield microscopy images across 30 individual cells per high-powered field judged to be representative of cells across multiple fields, *, p < 0.05; **, p < 0.01; ***, p < 0.001.

Supplemental Figure 4: Both volume and glucose uptake loss are MAPK-specific. 72h treatment with the MEK inhibitor AZD-6244 causes both A, volume loss and B, decrease in 2-NBDG uptake comparable to vemurafenib. Cells growth arrested by being cultured at 100% confluence for 72h did not display any significant decrease in C, 2-NBDG uptake or D, cellular volume.

Supplemental Figure 5: Brief exposure to hypotonic conditions does not cause significant cell death or membrane perturbation.

Supplemental Figure 6: Cap-dependent protein translation is significantly inhibited by vemurafenib by 72 hours of treatment. A, Immunoprecipitation of members of the eIF4F complex using m7 cap-bound sepharose beads shows decreased levels of eIF4e and eIF4g and increased levels of the inhibitor molecule 4EBP1. B, 35S-labeled methionine incubation of YUMAC cells treated with 3μM vemurafenib for increasing lengths of time reveals a decrease in de novo synthesis of hexokinase II that mirrors the downregulation observed by Western blot.

Supplemental Figure 7: Loss of translational activity involves initial downregulation of Akt/mTOR signaling upstream of eIF4F complex assembly. Cells were treated with 3μM vemurafenib or vehicle and harvested at labeled timepoints.

Supplemental Figure 8: Downregulation of Akt signaling upstream of eIF4F complex assembly mirrors early loss of S6 and p70S6K activity by 72 hours in sensitive but not resistant lines.. Cells were treated with 3μM vemurafenib (+) or DMSO vehicle (-) before harvesting and Western Blot.

Supplemental Figure 9: Inhibition of proteosomaldegradation is insufficient to rescue HKII loss.