Fig 1: Protein immunoblot analysis. Immunoblotting of the proteins involved in mitochondrial and metabolic functions was performed in the hippocampi from the control, depression-susceptible, anxiety-susceptible, and insusceptible groups.Por, Idh2, Esd, Glo1, G6pdx, Aldh2, Dld, Dlat, Ogdhl, Anxal, Tpp2, Sdha, Prnp, Pkm, and Prdx6 were detected with their respective antibodies. Each blot corresponded to the five rats used in the analysis, and Coomassie blue staining was used as the loading control. Dep-Sus depression-Susceptible, Anx-Sus Anxiety-susceptible, Insus Insusceptible, Cont Control; n = 5, *p < 0.05, **p < 0.01
Fig 2: Immunohistochemical evaluation of CYP1B1 and CPR colocalization with NeuN in hippocampal subregions. Confocal images depicting an overview of CA1, CA2 and CA3 at 10× magnification (a–c), subregion CA2 captured at 40× magnification (d–i). Green channel (a,d,g) depicts NeuN staining, red channel (b,e,h) CPR or CYP1B1 and (c,f,i) the two channels merged. Distinct colocalization of CPR and NeuN was observed in subregions CA1 and CA2, but not CA3 (c). Positive colocalization of CYP1B1 and NeuN was noted in several cells, indicating presence of CYP1B1 protein in CA2 neurons (i). Examples of cells judged as positive for CPR or CYP1B1 are marked by arrows. The image was deemed representative of all groups, as no apparent difference was observed between sexes, interventions, or survival times. Abbreviations: CA2: Cornu Ammonis 2, CPR: Cytochrome P450 reductase, CYP1B1: Cytochrome P450 1B1.
Fig 3: Hepatic TGFßr1 deletion repressed LPS/D-GalN–induced ferroptosis. (A and B) Immunohistochemistry staining of and 4-HNE in livers (right) of TGFßr1?hep-CKO and TGFßr1?hep mice after vehicle or LPS/D-GalN treatment and quantitative results (left). (C) Serum levels of Fe2+ (n = 6) in TGFßr1?hep-CKO and TGFßr1?hep mice after LPS/D-GalN treatment. (D) MDA assay of liver homogenates from TGFßr1?hep-CKO and TGFßr1?hep mice after LPS/D-GalN treatment (n = 4). (E and F) Western blots of GPX4 expression in TGFßr1?hep-CKO and TGFßr1?hep mice liver and quantitative result of GPX4/GAPDH (n = 6). (G) Representative Western blots of DHODH, FSP1, xCT, GPX4, and TFR expression in TGFßr1?hep-CKO and TGFßr1?hep mice liver and (H) ratios of each protein to GAPDH (n = 6). (I) Representative western blots of CHAC1 and POR expression in TGFßr1?hep-CKO and TGFßr1?hep mice liver and (J) ratios of each protein to GAPDH (n = 6). All data are presented as means ± SEM. Scale bar: 100µm. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. D-GalN, D-galactosamine; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Fig 4: DFOM alleviated LPS/D-GalN–induced ALF by inhibiting ferroptosis. (A) The survival rate of WT mice treated with vehicle or DFOM (100 mg/kg) 1 hour before LPS/D-GalN co-injection (n = 5). (B) Serum levels of ALT and AST from WT mice with vehicle or DFOM before LPS/D-GalN co-injection (n = 5). (C) Serum of Fe2+ levels of WT mice with vehicle or DFOM before LPS/D-GalN co-injection (n = 5). (D) Representative pictures for H&E and TUNEL staining of WT mice with vehicle or DFOM before LPS/D-GalN co-injection (n = 5). (E and F) GSH and MDA assay of liver homogenates from WT mice with vehicle or DFOM before LPS/D-GalN treatment (n = 5). (G and H) Western blot analyses of CHAC1, POR, TFR, and Ptgs2 from WT mice with vehicle or DFOM before LPS/D-GalN treatment and ratios of each protein to glyceraldehyde-3-phosphate dehydrogenase (GAPDH) (n = 6). (I and J) Western blot analyses of DHODH, FSP1, GPX4, and XCT from WT mice with vehicle or DFOM before LPS/D-GalN treatment and ratios of each protein to GAPDH (n = 6). All data were obtained from WT mice. Scale bars: 100 µm. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. D-GalN, D-galactosamine.
Fig 5: LPS/D-GalN-induced ALF enhances apoptosis and ferroptosis. (A) The survival rate of WT mice treated with vehicle or LPS/D-GalN (LPS, 20 µg/kg; D-GalN, 700 mg/kg) (n = 10). (B) Representative pictures of WT mice treated with vehicle or LPS/D-GalN for 3, 5, and 6 hours (n = 10). (C) Serum levels of ALT and AST with or without LPS/D-GalN co-injection (n = 6). (D) H&E and TUNEL staining after LPS/D-GalN co-injection (n = 6). (E) Immunohistochemistry of TNFa and F4/80 staining after LPS/D-GalN co-injection (n = 6). (F) Transmission electron microscope of liver tissues (n = 3). (G) MDA assay of liver homogenates (n = 5). (H and J) Western blot analyses of TFR, DMT1, GPX4, and XCT from WT mice (H) with or without LPS/D-GalN and (I) quantitative results (n = 3–9). (I and K) FSP1, DHODH, and POR proteins. Western blot analyses of WT mice (I) with or without LPS/D-GalN and (K) quantitative results (n = 3–9). (L) Immunohistochemistry of DMT1 staining and immunofluorescence of Ptgs2 in livers with or without LPS/D-GalN (n = 6). (M) Quantitative results of DMT1 and Ptgs2. All data were obtained from WT mice. Scale bars: 100 µm. Data are presented as means ± SEM. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. DAPI, 4',6-diamidino-2-phenylindole; GAPDH, glyceraldehyde-3-phosphate dehydrogenase.
Supplier Page from Abcam for Anti-Cytochrome P450 Reductase antibody [EPR14479(B)]