FIGURE 3.

Supplementation of BH4 protects CRC cells from ferroptosis promoted by GCH1 knockdown. (A) Supplementation of 50 µM BH4 restored the BH4 levels in siGCH1 transfected cells. (B-F) BH4 supplemented in (A) reversed cell death, lipid peroxidation, and ferrous iron accumulation caused by GCH1 knockdown during erastin treatment. (B) Cell viability was assessed by CCK-8. Lipid peroxidation was detected using the MDA assay kit (C) or BODIPY-C11 staining (D) (The ratio of lipid ROS normalized to erastin-treated siNC cells). The cellular Fe²⁺ (E) and mitochondrial Fe²⁺ (F) were detected using FerroOrange and MitoferroGreen staining, separately. Scale bar, 20 µM. The error bars represent standard deviation from at least three replicates (#p < 0.05, ##p < 0.01, ###p < 0.001, ####p < 0.0001, ns, not significant, compared between siNC and siGCH1 by unpaired t-test) (*p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, ns, not significant, compared between the two groups by unpaired t-test).