Fig 1: Influence of z-VAD-fmk, Nec-1, and BafA1 on apoptotic, necrotic, and autophagy signaling proteins, respectively. MIA PaCa-2 cells were pretreated with 5 μM z-VAD-fmk, 20 μM Nec-1, or 2 nM BafA1 for 2 h and were then treated with 0.25 μM GEM + 0.5 μM Pita for 48 h. (A–C) Cell lysates were analyzed using Western blot with the indicated antibodies. Quantitative data are presented as mean ± SD from three replicates. (A) Expression levels of the antiapoptotic marker Bcl-2 were reduced by GEM combined with Pita but were elevated by z-VAD-fmk. Meanwhile, the apoptotic markers of cleaved caspase-3 and cleaved PARP-1 were also suppressed by z-VAD-fmk. (B) Expression levels of the necrotic markers RIP3, RIP1, and MLKL were elevated by GEM combined with Pita but were suppressed by Nec-1. (C) Expression level of the autophagy marker LC3-II was elevated by GEM combined with Pita but was suppressed by BafA1. (D) MIA PaCa-2 cell viability was determined using CCK-8. Cells with GEM combined with Pita treatment exhibited significantly decreased cell viability. When pretreated with z-VAD-fmk, Nec-1, or BafA1, the cells exhibited increased cell viability. The density for the control group was set at 1; * and † represent significant difference p-values, P ˂ 0.05, compared to the untreated or G0.25+P0.5-treated group; mean ± SD from three replicates.Abbreviations: z-VAD-fmk, Z-Val-Ala-Asp-fluoromethylketone; Nec-1, necrostatin-1; Baf-A1, bafilomycin A1; GEM, gemcitabine; Pita, pitavastatin; Bcl-2, B-cell lymphoma 2; RARP-1, poly (ADP-ribose) polymerase 1; RIP1, receptor-interacting serine-threonine kinase 1; RIP3, receptor-interacting serine-threonine kinase 3; MLKL, mixed lineage kinase domain-like; LC3, light chain 3. CCK-8, cell counting kit-8.