Figure 4 (See previous page).

RIPK1 protects melanoma cells from TM- or TG-induced apoptosis by activation of autophagy. (A) Whole cell lysates from Mel-RMu cells stably transfected with pCMV-MYC or pCMV-MYC-RIPK1 were subjected to western blot analysis of RIPK1 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (B) Mel-RMu cells stably transfected with pCMV-MYC or pCMV-MYC-RIPK1 were treated with tunicamycin (TM) (3 μM) or thapsigargin (TG) (1 μM) for 16 h. Whole cell lysates were subjected to western blot analysis of SQSTM1, MAP1LC3A and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (C) Mel-RMu cells stably transfected with pCMV-MYC or pCMV-MYC-RIPK1 were treated with TM (3 μM) or TG (1 μM) for 48 h with or without pretreatment with bafilomycin A₁ (Baf A1) (10 nM) for 1 h. Cell viability was measured by CellTiter-Glo assays (n = 3, mean ±SEM, *P < 0.05, Student t test). (D) Mel-RMu cells stably transfected with pCMV-MYC or pCMV-MYC-RIPK1 were transiently transfected with the control or ATG5 siRNA followed by treatment with TM (3 μM) or TG (1 μM) for 48 h. Cell viability was measured by CellTiter-Glo assays (n = 3, mean ±SEM, *P < 0.05, Student t test). (E) Mel-RM (left panel) and MM200 (right panel) cells transduced with the control or RIPK1 shRNA were treated with TM (3 μM) or TG (1 μM) for 48 h with or without pretreatment with Baf A1 (10 nM) for 1 h. Cell viability was measured by CellTiter-Glo assays (n = 3, mean ±SEM, *P < 0.05, Student t test). (F) Mel-RM and MM200 cells transduced with the control or RIPK1 shRNA were treated with TM (3 μM) (upper panel) or TG (1 μM) (lower panel) with or without pretreatment with Baf A1 (10 nM) for 1 h. Whole cell lysates were subjected to western blot analysis of MAP1LC3A and GAPDH (as a loading control). The data shown are representative of 3 individual experiments. (G) Whole cell lysates from Mel-RM and MM200 cells treated with TM (3 μM) or TG (1 μM) for 16 h with or without pretreatment with Baf A1 (10 nM) for 1 h were subjected to western blot analysis of RIPK1 and GAPDH (as a loading control). The data shown are representative of 3 individual experiments.