Figure 6
Knocking down HO-1 but not ferritin heavy chain aggravates HIER stress in EC cultures. HO-1 and ferritin heavy chain 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) CHOP, (B) Grp78, and (C) HO-1 mRNAs as well (D) spliced XBP1, CHOP, HO-1, and ferritin heavy chain protein levels were analyzed 3 h after the heme treatment. In another set of experiments, relative expression of (E) CHOP, (F) Grp78, and (G) HO-1 mRNAs as well (H) Grp78, spliced XBP1, CHOP, HO-1, and ferritin heavy chain protein levels were analyzed 16 h after the heme treatment. (I) HO-1 was knocked down or upregulated with heme arginate as described above. 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 and HO-1 as well as Grp78 expressions were analyzed with immunofluorescence using super-resolution microscopy. GM growth medium, NC non-coding siRNA, HA heme arginate, PC positive control. Relative mRNA expressions were normalized to GAPDH. GAPDH was used as a loading control in immunoblots. Immunoblots are cropped from different parts of the same gel. Uncropped immunoblots are presented in the Supplementary information. Data are shown as mean ± SEM of three independent experiments. Statistical analysis was performed by one-way ANOVA test followed by Bonferroni correction. A value of p < 0.05 was considered significant. ***p < 0.001.
