Figure 4

ER stress inhibitors does not protect against HIER stress. ECs were pre-incubated with either 4-phenylbutyric acid (4-PBA) (5 mM) or valproic acid (VPA) (5 mM) overnight, then exposed to heme (25 µM) in serum- and antibiotics-free CM199 supplemented with either 4-PBA (5 mM) or VPA (5 mM), then further incubated with CM199 supplemented with 10% FCS, antibiotics, and either 4-PBA (5 mM) or VPA (5 mM) for 3 h and 6 h. (A) Relative expression of CHOP, (B) Grp78, and (C) HO-1 were analyzed after 3 h and (E) CHOP, (F) Grp78, (G) HO-1 6 h. Representative immunoblots of three independent experiments are shown representing Grp78, spliced XBP1, CHOP, HO-1, and ferritin heavy chain levels after (D) 3 and (H) 6 h. GM growth medium, VC vehicle control, PC positive control. Data are shown as mean ± SEM of three independent experiments. Immunoblots are cropped from different parts of the same gel. Uncropped immunoblots are presented in the Supplementary information. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni correction. A value of p < 0.05 was considered significant. *p < 0.05, *p < 0.01, ***p < 0.001.