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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 proteosomal degradation is insufficient to
rescue HKII loss.