Fig. 5: Influence of HO-1 on hemin-triggered ROS and oxidative DNA damage in HCEC and CRC cells.

a HCEC were incubated with hemin (0 or 50 µM) in the absence or presence of the HO-1 inhibitor zinc protoporphyrin (ZnPP; 0.5 or 1 µM) for 24 h. Cells were stained with the CM-H2DCFDA dye and levels of reactive oxygen species (ROS) were assessed by flow cytometry. Data are presented as mean + SEM (n ≥ 3). *p < 0.05; **p < 0.01; ***p < 0.001. b HCT116 cells were treated and analyzed as described above. Data are given as mean + SEM (n = 4). *p < 0.05; **p < 0.01; ****p < 0.0001. c HCEC and HCT116 cells were transiently transfected with scrambled (scr) or HO-1-specific siRNA. 24 h after transfection, cells were treated with hemin and incubated for another 24 h. Whole-cell extracts were analyzed by SDS-PAGE and immunoblot detection of Nrf2 and FtH. Hsp90 served as loading control, whereas HO-1 was visualized to confirm knockdown. d Knockdown of HO-1 in HCEC followed by hemin exposure for 24 h and analysis of ROS formation. ROS were measured by flow cytometry as stated above. Data are given as mean + SEM (n = 4). *p < 0.05; ****p < 0.0001. e HCEC were treated with hemin (0 or 50 µM) in the absence or presence of the HO-1 inhibitor ZnPP (ZnPP; 0.5 or 1 µM) for 24 h. Cells were then subjected to the alkaline Comet assay with or without Fpg. OTM, olive tail moment. Data are given as mean + SEM (n = 5). Ns: p > 0.05; *p < 0.05; **p < 0.01; ***p < 0.001, ****p < 0.0001. f Representative pictures of the data shown in e.