Figure 2

PAC-1 induces UPR and ER stress in multiple cell types including apoptotic defective cell lines. (a) Western blot demonstrates that PAC-1 did not alter the expression of proteins XIAP, Bak, Bak, Bcl-2, Hsp27, Hsp70 and Hsp90 significantly but it could induce significant upregulation of ER stress marker GRP78 and GRP94 proteins in both MCF7 and MCF7C3 cells. β-Actin was used as loading control. (b) PAC-1 induced expression of several UPR indicator proteins such as CHOP, IRE1α, Ero1α and p-eIF2α both in MCF7 and MCF7C3 cells. β-Actin was used as loading control. (c) PAC-1 treatment upregulated ER stress marker protein GRP78 in multiple cancer cell lines such as MCF7, HCT116, U2OS, SKOV3, SiHa and HeLa. β-Actin was used as loading control. (d) Splicing of XBP1 mRNA was observed by RT-PCR in multiple cancer cells such as MCF7, HCT116, SiHa and Hela after PAC-1 treatment. Thapsigargin was used as positive control. (e) Cell death was analyzed by annexin V binding and PI staining in MEF WT and Bax/Bak DKO cells using FACS. The percentages of PI and annexin-positive cells are mentioned in Q2 quadrant representing apoptotic cells. Western blot and RT-PCR results suggest that PAC-1 induced upregulation of GRP78 as well as splicing of XBP1 mRNA in both MEF WT and DKO cells. β-Actin was used as loading control. (f) Cell death was analyzed by PI staining in MEF WT and MEF Apaf-1 KO cells using FACS after PAC-1 (50 μM) treatment. There was negligible cell death in Apaf-1 KO cells than its WT counterpart. The percentage of PI-positive cells is represented in P2 gate. Furthermore, western blot and RT-PCR results suggest that PAC-1 induced splicing of XBP1 mRNA and upregulation of GRP78 efficiently in WT as well as Apaf-1 KO cells even though cell death was absent in Apaf-1 KO cells