Fig. 5: Elevated ROS levels drive RIP kinase activation and MPTP opening.

SK-HEP-1 cells were treated for 24 h with the control, TNF-α (25 ng/mL), bicarbonate (60 mM), or a combination (TNF-α + bicarbonate) in the presence or absence of NAC (1 mM) or EUK8 (20 μM). a Representative images of MitoSOX Red (mitochondrial ROS) and DCFH-DA (cytosolic ROS) fluorescence. Scale bar: 20 µm. The data are presented as the means ± SDs (n = 3 biological replicates). b NAC and EUK8 suppressed mitochondrial ROS production induced by bicarbonate + TNF-α. The data are presented as the means ± SDs (n = 3 biological replicates). c Western blot analysis of the levels of pRIPK1, pRIPK3, pMLKL and total proteins, and the quantification of the ratios of the phosphorylated proteins to the total proteins. The data are presented as the means ± SDs (n = 3 biological replicates). d, e Representative images of immunofluorescence staining showing NF-κB (p65) nuclear translocation. Red arrows indicate p65-positive nuclei. Quantification of nucleus-localized NF-κB. Scale bar: 20 µm. The data are presented as the means ± SDs (n = 3 biological replicates). f ROS inhibitors prevented the MPT (calcein-AM). The data are presented as the means ± SDs (n = 3 biological replicates). g ROS inhibitors attenuated Δψm depolarization (TMRE). The data are presented as the means ± SDs (n = 3 biological replicates). h ROS inhibition rescued cell viability. The data are presented as the means ± SDs (n = 3 biological replicates). Unpaired t test. ns, P > 0.05; *P < 0.05; **P < 0.01; and ***P < 0.001.