Fig. 5: Decreased NAD⁺ levels activated the FoxO1 phosphorylation to inhibit GPX4 expression and promote ferroptosis.

A Design of luciferase reporter vector containing human GPX4 promoter with a FoxO1 binding site (pro-GPX4) or a mutant FoxO1 binding site (pro-GPX4-mut). The red indicates mutant bases. B ChIP assay using IgG or FoxO1 antibody and quantification of the enrichment of FoxO1 binding to GPX4 promoter in HeLa cells. C ChIP assay using FoxO1 antibody and quantification of the enrichment of FoxO1 binding to GPX4 promoter in Parental (n = 1), MTCH1^WT (n = 2), and MTCH1^KO (n = 2) HeLa clones. D Luciferase reporter assay of FoxO1 binding to GPX4. The FoxO1 binding site was shown in (A). E Immunoblots of lysates from Parental (n = 1), MTCH1^WT (n = 2), and MTCH1^KO (n = 2) HeLa clones with the indicated antibodies. F Immunoblots of lysates from the nuclear and cytoplasmic (non-nuclear) of Parental (n = 1), MTCH1^WT (n = 2), and MTCH1^KO (n = 2) HeLa cell clones with the indicated antibodies. G Quantitative analysis of FoxO1 in the nucleus (F) relative to Histone. H Representative fluorescence imaging (n = 6 images in each group) of FoxO1 (red) and nucleus (blue) in MTCH1^WT and MTCH1^KO HeLa clones. Scale bar, 100 µm. I, J The mean fluorescence intensity (MFI) of nuclear FoxO1 (I) and the nuclear translocation percentage of FoxO1 (J) in MTCH1^WT and MTCH1^KO HeLa clones. K Immunoblots of lysates from Parental (n = 1) and MTCH1^KO (n = 3) HeLa clones treated with or without 5 mM NAD⁺ with the indicated antibodies. L Immunoblots of lysates from MTCH1^KO HeLa clones treated with 5 mM NAD⁺ for different time (0, 6, 12, 18, 24 h) with the indicated antibodies. M, N Transcriptional levels of GPX4 (M) and ROS content (N) in HeLa cells transfected with sh-scramble or sh-FoxO1 plasmid. O Transcriptional levels of GPX4 in MTCH1^WT (n = 2) and MTCH1^KO (n = 3) HeLa clones treated with DMSO, FoxO1 activator LOM612 or FoxO1 inhibitor AS1842856. P Immunoblots of lysates from Parental (n = 1), MTCH1^WT (n = 2), and MTCH1^KO (n = 3) HeLa clones transfected with FoxO1-AAA plasmid or not with indicated antibodies. Q–T Relative cell viability (Q), GPX4 transcriptional levels (R), ROS content (S) and mitochondrial MDA content (T) in Parental (n = 1), MTCH1^WT (n = 2), and MTCH1^KO transfected with FoxO1-AAA plasmid or empty vector (n = 3) HeLa clones. Data were presented as mean ± SEM of at least three independent replicates (*P < 0.05, **P < 0.01, ***P < 0.001) and analyzed by one-way ANOVA with Tukey’s multiple comparisons test or unpaired t-test.