Figure S3. DUSP4-induced ERK hyperactivation is the major event leading to oncogenic overdose phenotype.

(A) SKMEL28 was transfected with siRNA against DUSP4 and nontargeting control and treated with trametinib at the indicated concentrations or with DMSO as a vehicle. Representative images after 5 d post-transfection are shown. (B) SKMEL28 and COLO829 were transfected cells as in (A) and were treated with cobimetinib at the indicated concentrations. The cell growth was analyzed by measuring cell confluence over time. Cell growth values were normalized against time 0. Data are mean ± SEM, n = 2. (C) SKMEL28 and COLO829 were transfected cells as in (A) and were treated with cobimetinib (Cobi) at the indicated concentrations or with DMSO as a vehicle (0 nM). Forty-eight hours later, lysates of the two melanoma cell lines were analyzed by immunoblot. Band intensities were analyzed by ImageJ software, and the P-ERK/GAPDH ratios are indicated in the histograms. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated against siNT or siDUSP4. (D) Cells were transfected cells as in (A) and were treated with a p38i (Skepinone-L) at the indicated concentrations or with DMSO as a vehicle (0 nM). The cell growth was analyzed by measuring cell confluence over time. Cell growth values were normalized against time 0. Data are mean ± SEM, n = 2. (E) Cells were treated with a p38i (Skepinone-L) at the indicated concentrations or with DMSO as a vehicle (0 nM). One hour later, cells were treated for 1 h with 100 μM H₂O₂, and cell lysates of both COLO829 and SKMEL28 were analyzed by immunoblot. Band intensities were analyzed by ImageJ software, and the P-HPS27/GAPDH ratios are indicated in the histograms. Data represent mean ± SEM of three independent experiments. Statistical significance was calculated against untreated cells or H₂O₂ treated cells.