Figure 4.

Knockdown of Ireb2 in SHPs can reduce the level of Fe²⁺, inhibit ferroptosis, and alleviate cell injury in SHP-HR. Evaluation of ferroptosis and cell injury in SHP-HR with si-NC or si-IREB2. (a) IREB2/FTH1/TFR1 protein levels assessed using western blotting. (b) Semiquantitative analysis of (a). (c) The Fe²⁺ level detected using colorimetry. (d) The MDA content detected using colorimetry. (e) The GSH content detected using colorimetry. (f) The Ptgs2 mRNA level. (g) The lipid ROS level detected using C11-BODIPY and flow cytometry. (h) Quantitative analysis of C11-BODIPY fluorescence intensity. (i) Cell viability detected using a CCK-8 kit. (j) Cell mitochondrial membrane potential detected using JC-1 staining and fluorescence microscopy, showing that the JC-1 monomer (green) increased and the polymer (red) decreased in HR with si-NC, representing mitochondrial membrane potential dysfunction, while si-IREB2 reversed this change and improved the mitochondrial membrane potential. (k) Cell death ratio detected using PI staining and flow cytometry. (l) Quantitative analysis of (k). n = 3 per group. Data are presented as the mean ± SEM. ^∗P < 0.05; ^∗∗P < 0.01.