Fig. 3.
In vitro validation of Feb-1 drug effectiveness. (a) The effects of Feb-1 and Fer-1 on cell viability of Huc-MSCs were identified using CCK-8 (n = 3, one-way ANOVA, Tukey's post hoc tests). The results indicate that the drug has no obvious effect on cell viability at drug concentrations below 1 μM. (b) Exploring the appropriate concentration of RSL3 to induce ferroptosis (n = 6, one-way ANOVA, Tukey's post hoc tests, ****P < 0.0001). (c–e) Rescue of H2O2-induced cell death in Huc-MSCs by different concentrations of Fer-1 and Feb-1. (n = 6, one-way ANOVA, Tukey's post hoc tests, ***P < 0.001, and ****P < 0.0001). (f) Rescue of RSL3-induced ferroptosis by Feb-1 and Fer-1 at 1 μM concentration. Ferroptosis induction was performed using a 6 μM concentration of RSL3 (n = 6, one-way ANOVA, Tukey's post hoc tests, *P < 0.05, and ****P < 0.0001). (g) Quantitative detection of intracellular GSH (n = 6, one-way ANOVA, Tukey's post hoc tests, ****P < 0.0001). (h–i) Quantification of intracellular ROS using DCFH-DA (green), scale bar = 200 μm (n = 12, one-way ANOVA, Tukey's post hoc tests, ****P < 0.0001). (j–l) Western blot analyzed the effect of Feb-1 and Fer-1 on RSL3-induced ferroptosis. Feb-1 rescued the RSL3-induced decrease in GPX-4 and xCT protein expression (n = 9, one-way ANOVA, Tukey's post hoc tests, *P < 0.05, **P < 0.01, ***P < 0.001, and ****P < 0.0001).