Figure 5

HSPA5 is deacetylated by HDAC6. (A) Western blot analysis of HDAC6 protein expression in PANC1 and SW1990 cells following treatment with RSL3 (0–1 μM) for 0–24 h. (B) Western blot analysis of HDAC6 protein expression in PANC1 and SW1990 cells following treatment with erastin (0–15 μM) for 0–24 h. (C) Immunoprecipitation (IP) analysis of the interaction between HSPA5 and HDAC6 following treatment with RSL3 (1 μM) for 12 h. (D) Western blot analysis of HDAC6 protein expression in HDAC6-knockdown PANC1 and SW1990 cells. (E–G) Measuring cell survival by cell count kit 8, cell death by PI/hoechst33342 staining in the indicated cells following treatment with RSL3 (1 μM) for 24 h. Scale bar: 100 μm. (H-I) Measuring lipid peroxidation and oxidative stress using an MDA assay kit or BODIPY C11 probe in indicated cells following treatment with RSL3 (1 μM) for 24 h. (J) Immunoprecipitation analysis of HSPA5 acetylation using anti-acety-lys treatment with TSA (0.1 mM) or RSL3 (1 μM) for 12 h. (K) Immunoprecipitation analysis of HSPA5 acetylation in HDAC6 knockdown PDAC cells. (L) Immunoprecipitation analysis of HSPA5 acetylation following treatment with RSL3 (1 μM) in HDAC6 knockdown PDAC cells. PDAC, pancreatic ductal adenocarcinoma; PI, propidium iodide; Ace-lys, acetylated lysine.