Figure S2.
SPRED1 inactivation confers resistance to BRAF inhibition in BRAF-driven human melanoma cell lines. (A) Viability of BRAF-driven UACC257 human melanoma cells stably expressing a control shRNA (shCTRL) or shRNAs directed against SPRED1 (shSPRED1#2 and #4) and treated with increasing concentrations of the BRAFV600E inhibitor dabrafenib for 4 d. Mean ± SD of three independent experiments; *, P < 0.05 for both shRNAs against SPRED1 compared with the control shRNA, paired t test. (B) Relative viability at 270 nM dabrafenib between A375 cells expressing shRNAs against SPRED1 (shSPRED1#2 and #4) and cells expressing a control shRNA in the presence (+SPRED1) or absence (−SPRED1) of inducible SPRED1 expression. Mean ± SD of four independent experiments. shSPRED1#2: *, P = 0.03; shSPRED1#4: *, P = 0.01, paired t test. (C) Western blot analysis of MAPK pathway activity in the cells described in A treated with the indicated concentrations of dabrafenib (Dab) for 6 h. C, shCTRL; S2, shSPRED1#2; S4, shSPRED1#4. Representative of three independent experiments. (D) Viability of the cells described in A treated with increasing concentrations of the MEK inhibitor trametinib for 4 d. Mean ± SD of three independent experiments; P > 0.05 (not significant), paired t test. (E) Western blot analysis of SPRED1, active Ras (Ras-GTP) and total Ras levels in BRAF-driven A375 human melanoma cells stably expressing a control shRNA (C) or shRNAs directed against SPRED1 (S2 and S4) and treated with the indicated concentrations of dabrafenib for 6 h. Signal intensity ratio of Ras-GTP to total Ras is indicated. Representative of two independent experiments. (F) Western blot analysis of MAPK pathway activity and SPRED1 levels in BRAF-driven A375 human melanoma cells treated with 100 nM dabrafenib (+dab) for the indicated durations before drug washout and further culture in the absence of treatment (−dab) for the indicated durations. Representative of three independent experiments.