FIGURE 11.

Heme-generated ROS is involved in heme-induced endoplasmic reticulum (ER) stress. Cells were treated with heme (25 μM) alone or heme (25 μM) + rA1M (12.5 μM) or heme (25 μM) + Hpx (25 μM) or heme (25 μM) + N-acetyl cysteine (NAC; 10 mM) as described for Figure 8 followed by the analysis of ROS generation and ER stress marker expression (A–F). (A) Heme induced ROS generation was assessed by CM-H2DCFDA assay. Results are presented as mean ± SD of five independent experiments. ^∗∗∗p < 0.0001. (B) Inhibition of heme-induced ROS generation by Hpx, rA1M and NAC using CM-H2DCFDA assay. Results are presented as mean ± SD of five independent experiments. ^∗∗∗p < 0.0001 (C–E) Relative expressions of CHOP, ATF4 and HO1 in response to heme and heme + NAC were determined by qRT-PCR, normalized to GAPDH. mRNA levels of heme + NAC treted cells were compared to those detected in heme challenged cells. Results are presented as mean ± SD of five independent experiments performed in duplicates. ^∗∗∗p < 0.0001 using unpaired t-test. (F) Representative Western blots of whole cell lysates from five independent experiments are shown representing ATF6 proteolysis, XBP1s expression, eIF2α phosphorylation and subsequent ATF4 induction as well as HO1 and FTH expression.