Figure 2
Heme activates ER stress in EC cultures in a time- and dose-dependent manner. ECs were treated with various doses of heme (10, 25, and 50 μM) or corresponding vehicle solution to the highest heme dose (50 μM) in serum- and antibiotics-free CM199 for 2 h, then the medium was changed to CM199 with 10% FCS and antibiotics. Thapsigargin (1 μM) treated cells were used as positive control. (A) Relative expression of CHOP and (B) Grp78 mRNA were analyzed after 3, 6, or 16 h and normalized to GAPDH. Representative immunoblots of three independent experiments are shown representing Grp78, spliced XBP1, CHOP, HO-1, and ferritin heavy chain levels (C) 3, (D) 6, and (E) 16 h after the heme treatments. 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 < 0.001.
