Fig 1: FSP1 reduces CoQ10 to prevent lipid peroxidation in intestinal epithelial cells (A–D) Levels and ratios of total, reduced, and oxidized CoQ10 in control and FSP1KO Caco-2 cells. n = 5, unpaired two-tailed t test; n.s., not significant. (E-H) Levels and ratios of total, reduced, and oxidized CoQ10 in Caco-2 cells treated with 5 µM iFSP. n = 5, unpaired two-tailed t test; n.s., not significant. (I-L) Levels and ratios of total, reduced, and oxidized CoQ10 in Caco-2 cells treated with 10 µM CoQ10. n = 5, unpaired two-tailed t test; n.s., not significant. (M−P) Levels and ratios of total, reduced, and oxidized CoQ10 in control and FSP1-overexpressing Caco-2 cells. n = 5, unpaired two-tailed t test; n.s., not significant. (Q) NADH consumption rate using recombinant purified human FSP1 combined with different oxidizing substrates. The panels are representative of three independent biological replicates. (R) Representative autoxidation inhibition by 100 nM recombinant human FSP1 and 10 μM CoQ10 or 10 μM idebenone with 1 μM STY-BODIPY and 25 μM NADH. The “uninhibited” group served as the negative control and did not contain CoQ10, showing maximal lipid peroxidation. The panels are representative of three independent biological replicates.
Fig 2: CoQ10 mitigates intestinal IRI via the FSP1-mediated reduction of CoQ10 (A) AAV9-shCtrl or AAV9-shFSP1 was injected intraperitoneally into intestinal tissue at multiple time points. EGFP expression in the mucosa, submucosa and muscle layer of the intestines was detected directly by fluorescence microscopy. Scale bar, 50 µm. (B) Schematic showing the chronological order of AAV9-mediated FSP1 knockdown, CoQ10 supplementation and intestinal IRI model establishment. (C) Representative H&E staining of intestines subjected to ischemia–reperfusion in the AAV9-shCtrl or AAV9-shFSP1 group with/without CoQ10 supplementation. Scale bar, 100 µm, n = 5. (D-E) Representative immunofluorescence staining of ZO-1 and Occludin in the intestines of mice subjected to ischemia–reperfusion in the AAV9-shCtrl or AAV9-shFSP1 groups with/without CoQ10 supplementation. Scale bar, 100 µm, n = 5. (F) Serum levels of I-FABP in mice in the AAV9-shCtrl or AAV9-shFSP1 group with/without CoQ10 supplementation. n = 5; two-way ANOVA, n.s., not significant. (G) Levels of ferrous iron in the IRI intestines of mice in the AAV9-shCtrl- or AAV9-shFSP1-treated group with/without CoQ10 supplementation. n = 5; two-way ANOVA, n.s., not significant. (H) MDA levels in the IRI intestines of the AAV9-shCtrl- or AAV9-shFSP1-treated mice with/without CoQ10 supplementation. n = 5; two-way ANOVA, n.s., not significant. (I) Oxidized and reduced CoQ10 in intestines subjected to IRI from AAV9-shCtrl- or AAV9-shFSP1-treated mice with/without CoQ10 supplementation. n = 5; two-way ANOVA, n.s., not significant.
Fig 3: CoQ10-mediated suppression of ferroptosis is dependent on FSP1 in intestinal epithelial cells (A) Caco-2 cells treated with 3 nM RSL3 or 3 nM RSL3 following the H/R procedure were labeled with BODIPY 581/591 C11 and fixed before imaging. Images are representative of at least 20 cells imaged for each treatment condition. Ox., oxidized; Non-ox., nonoxidized. Scale bar, 50 µm. (B) Representative live-dead staining images; live-dead staining was used to analyze the viability of Caco-2 cells treated with 3 nM RSL3 or 3 nM RSL3 following H/R. Scale bar, 50 µm. (C) Dose response analysis of RSL3-induced cell death in control Caco-2 cells and pretreated Caco-2 cells subjected to H/R. (D) Caco-2 cells were treated with 15 nM RSL3 and 50 nM Lip-1, 25 µM DFO or 10 nM CoQ10 for 12 h, labeled with BODIPY 581/591 C11 and fixed before imaging. Images are representative of at least 20 cells imaged for each treatment condition. Ox., oxidized; Non-ox., nonoxidized. Scale bar, 50 µm. (E) Representative live-dead staining images; live-dead staining was used to analyze Caco-2 cells treated with 15 nM RSL3 and 50 nM Lip-1, 25 µM DFO or 10 µM CoQ10 for 12 h. Scale bar, 50 µm. (F) Dose response analysis of RSL3-induced cell death in the presence of ferroptosis inhibitors. Lip-1, 50 nM; DFO, 25 µM; CoQ10, 10 µM. (G) Dose response analysis of RSL3-induced cell death in control and FSP1KO Caco-2 cells. (H-I) Dose response analysi of RSL3-induced cell death in control and FSP1KO Caco-2 cells in the presence of 4-CBA or 4-CBA and CoQ10. 4-CBA, 3 mM; CoQ10, 10 µM. (J-K) Dose response analysis of RSL3-induced cell death in the indicated Caco-2 cell lines treated with 10 µM CoQ10. C, F-K, shading indicates 95 % confidence intervals for the fitted curves, and each data point is the average of three technical replicates. The panels are representative of three biological replicates.
Fig 4: FSP1 suppresses ferroptosis by preventing phospholipid peroxidation in intestinal epithelial cells (A) Volcano plot showing the average fold changes and P values of phospholipid species in control and FSP1KO Caco-2 cells treated with 15 nM RSL3 under culture conditions including supplementation with 10 µM CoQ10. n = 4. PE, phosphatidylethanolamine; PC, phosphatidylcholine; LPE, lysophosphatidylethanolamine; LPC, lysophosphatidylcholine; O-, ether-linked phospholipid. (B) Plot showing the average fold changes of representative phospholipid species in control and FSP1KO Caco-2 cells treated with 15 nM RSL3 under culture conditions including supplementation with 10 µM CoQ10. n = 4. (C-Q) Levels of representative unsaturated phospholipids, lysophospholipids and ether-linked phospholipids in control and FSP1KO Caco-2 cells treated with 15 nM RSL3 under culture conditions including supplementation with 10 µM CoQ10. n = 4, unpaired two-tailed t test.
Supplier Page from Sino Biological, Inc. for Mouse AIFM2/AMID Gene ORF cDNA clone expression plasmid