Figure 9. ROS involved in P53-mediated mitochondrial apoptotic pathway.

HUVEC cells were pretreated with or without MnTMPyP (10 umol/l) for 1 h before exposure to heat stress. (a) the effect of MnTMPyP on the production of ROS in HUVEC cells were assayed by DCFH staining.(b)Flow cytometry analysis of the effect of MnTMPyP on heat stress-induced cells apoptosis. H₂O₂ was used as a positive control. (c)The effect of MnTMPyP on enzymatic activity of caspase-9 was measured in cell lysates using fluorogenic substrate Ac-LEHD-AFC and was expressed relative to the control at 37°C (100%). (d) Flow cytometry and JC-1 to measure the effect of MnTMPyP on ΔΨm. (e–f): Westen blots analysis(cropped) on the effects of MnTMPyP on P53 mitochondrial translocation, and cytochrome c release from mitochondria induced by intense heat stress. COX IV, mitochondrial loading control. Data are presented as mean ± SD for three independent experiments, *P < 0.05 compared with HS group.