Fig. 6: AIF, but not AIF3, possesses hitherto uncharacterized GR activity and regulates glutathione regeneration for ROS clearance.

a Scheme of three main ROS clearance pathways and a proposed role of AIF in ROS clearance. b Effects of AIF3 on the expression of key enzymes in ROS clearance pathways. n = 3 biological replicates. c–e Metabolomics analysis of GSH/GSSG in AIF3 splicing MEFs. n = 5 biological replicates. f GR activity in the presence of 3 μM purified AIF, AIF3, or AIF3L. n = 17 (GR/GR + AIF/GR + AIF3), and 14 (GR + AIF3L) biological replicates. g Structural comparison of AIF (PDB 1gv4) and GR (PDB 1ger). C, C-terminal domain. h GR activity of 3 μM purified AIF and AIF3 in time-dependent manner. n = 3 (Auto/AIF3), and 6 (AIF) biological replicates. i GR activity of purified AIF, AIF3 (3 μM), and GR (0.1 mU). n = 7 (Auto), 6 (AIF), 3 (AIF3), and 4 (GR) biological replicates. j Expression of AIF/AIF3 and GR proteins in AIF3 splicing, AIF KO, GR KO, AIF-GR DKO, and DKO/AIF(3) rescue MEFs. k, l Effects of AIF and AIF3 on GR activity in cells established in (j). n = 6 biological replicates. m GR activity in the forebrain of WT and AIF3 splicing mice (Emx1-Cre + ). n = 12 (WT) and 16 (AIF3) biological replicates (unpaired 2-tailed Student’s t test). n GR activity of purified mouse AIF (mAIF) proteins and its mutants (3 μM). n = 7 biological replicates. o Analysis of mitochondrial ROS levels in AIF3 splicing, AIF KO and AIF-GR DKO MEFs treated with DMSO or H₂O₂ in the absence or presence of GSH. n = 9 (WT/AIF3) and 6 (AIFKO/DKO) biological replicates (two-way ANOVA with Sidak’s multiple comparison test). Data are shown as mean ± S.E.M throughout this figure. One-way ANOVA with Tukey’s multiple comparison test was applied in c–f, i, k, l, n.