Fig. 3.

The EIF2AK3/PERK‐dependent ER‐stress response upregulated IL24 expression. (A) Melanoma A375 cells were cultured with either vehicle (V), 0.05 μm WX8, or 1 μm WX8 for either 24 or 48 h. Whole cell extracts were then immuno‐blotted for the indicated proteins, which were identified by their molecular weight and their reaction with a specific antibody. Thapsigargin (THAP) 1 μm was included for comparison. (B) Cells were treated for 24 h with vehicle (V), 0.05 μm WX8 or 1 μm WX8 and the indicated proteins identified by immuno‐blotting from both cytoplasm and nuclear lysate. Lamin B1 (LMNB1) and Vinculin (VCL) were used as nuclear and cytoplasmic control proteins. (C) Cells were treated for 48 h with vehicle (V), 1 μm WX8 or 1 μm THAP, and either nontargeted siRNA (nt‐siRNA) or siRNA targeted against DDIT3 (siDDIT3). The indicated proteins were detected by immunoblotting whole cell extracts. (D) Cells were cultured for 24 h with increasing concentrations of WX8, and the indicated proteins (eIF2α, eIF2α‐P, and ACTB) identified by immunoblotting of whole cell extracts. (E) IL24 protein [both glycosylated and de‐glycosylated (PNGase F treated) were immunoblotted from samples as described in panel D]. (F) The ratios of IL24/ACTB and EIF2A‐P/ACTB were quantified by densitometry from the data in panels D and E. (G) The ratio of IL24 RNA relative to HFF1 IL24 RNA was determined by RT‐PCR in the indicated cell lines (± SEM, n = 3) and compared with cell viability, as previously quantified by their IC₅₀ for ATP loss [13]. (H) The indicated cell lines were cultured for 24 h with vehicle, 0.05 μm WX8 (gray bar) or 1 μm WX8 (black bar). The ratios of IL24 RNA in WX8‐treated cells to vehicle‐treated cells were quantified by RT‐PCR (±SD, n = 2). Images in panels A through F are representative of three samples.