Figure 9
Knocking down biliverdin reductase does not aggravate HIER stress in EC cultures. HO-1 and biliverdin reductase A expressions were knocked down with small interfering RNAs, then ECs were exposed to heme (10 μM) for 2 h in serum- and antibiotics-free CM199. Then, the medium was changed to CM199 with 10% FCS and antibiotics. Relative expression of (A) BVRA, (B) CHOP, and (C) Grp78 mRNAs as well (D) spliced XBP1, CHOP, HO-1, and BVRA protein levels were analyzed 3 h after the heme treatment. In another set of experiments, BVRA was knocked down, then, ECs were treated with heme arginate as described above. Then, ECs were exposed to heme (25 μM) for 2 h in serum- and antibiotics-free CM199. Then, the medium was changed to CM199 with 10% FCS and antibiotics. Relative expression of (E) BVRA, (F) CHOP, and (G) Grp78 mRNAs as well (H) spliced XBP1, CHOP, HO-1, and BVRA protein levels were analyzed 3 h after the heme treatment. 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.
