ACSL4/FACL4 Antibody - #DF12141

Fig. 1. Renal tubular epithelial cells undergo ferroptosis in I/R-AKI. (A) Single-cell sequencing identified clusters in both sham-operated and I/R-operated kidneys at day 3 post-surgery, depicted in a t-distributed stochastic neighbor embedding (tSNE) map. PT, proximal tubule; DT, distal convoluted tubule; LOH, ascending loop of Henle; ECs, endothelial cells. (B) Average expression levels of mouse single cell type-specific genes are shown in pheatmap. Mean expression values of the genes were calculated in each cluster. The full list of cell types and genes is shown in Additional file1: Table S1. (C-E) Subtypes of PT with differentially expressed genes are illustrated in a tSNE plot (C), and the representative marker genes across the 3 subtypes of PT are depicted in pheatmap (D) and VlnPlot (E). (F-G) The expression levels of ferroptosis-associated genes Gpx4 and Acsl4 in the subtypes of PT were identified by FeaturePlot (F) and Dotplot (G). (H-I) Western blot (H) and statistical analysis (I) of the expression levels of GPX4 and SLC7A11 proteins in the renal cortex of sham-operated and I/R-operated mice post-surgery. ***p 

Figure 3 Deoxycholic acid enhanced lipopolysaccharide-induced ferroptosis in intestinal epithelial cells (A–H) IEC-6 cells and Caco-2 cells were incubated with or without 10 ng/mL LPS for 12 h, followed by 200 μM DCA for 2 h. They were divided into four group (n = 6 in each group): control group, DCA group, LPS group and LPS + DCA group. (A) The relative mRNA expression level of GPX4 in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (B) The relative mRNA expression level of ACSL4 in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (C) The protein level of GPX4, ACSL4 and β-actin in each group of IEC-6 cells and Caco-2 cells. (D) Representative transmission electron microscopy images (scale bars, 1 μm) of cell and mitochondria, and Fe2+/Hoechst immunofluorescence in each group of IEC-6 cells. Mitochondria was showed by white arrows in the pictures. The red fluorescence is indicative of the presence of ferrous ions. (E) The content of Fe2+ in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (F) The content of GSH in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (G) The content of ROS in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (H) The content of MDA in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (I-K) IEC-6 cells were incubated 2 μM ferrostatin-1 (Fer-1) for 16 h, and then incubated with or without 10 ng/mL LPS for 12 h, followed by 200 μM DCA for 2 h. They were divided into six group (n = 5 or 6 in each group): control group, LPS group, LPS + DCA group, Fer-1 group, LPS + Fer-1 group and LPS + DCA + Fer-1 group. (I) The relative mRNA expression level of GPX4, ACSL4 and DMT1 in each group of IEC-6 cells (n = 6 in each group). (J) The content of GSH in each group of IEC-6 cells (n = 5 in each group). (K) The content of MDA in each group of IEC-6 cells (n = 5 in each group). Data are represented as mean ± SEM. ns P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. ##P < 0.01, ####P < 0.0001. ∗ LPS group vs control group, LPS + DCA group vs LPS group. # LPS + Fer-1 group vs LPS group, LPS + DCA + Fer-1 group vs LPS + DCA group.

Figure 3 Deoxycholic acid enhanced lipopolysaccharide-induced ferroptosis in intestinal epithelial cells (A–H) IEC-6 cells and Caco-2 cells were incubated with or without 10 ng/mL LPS for 12 h, followed by 200 μM DCA for 2 h. They were divided into four group (n = 6 in each group): control group, DCA group, LPS group and LPS + DCA group. (A) The relative mRNA expression level of GPX4 in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (B) The relative mRNA expression level of ACSL4 in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (C) The protein level of GPX4, ACSL4 and β-actin in each group of IEC-6 cells and Caco-2 cells. (D) Representative transmission electron microscopy images (scale bars, 1 μm) of cell and mitochondria, and Fe2+/Hoechst immunofluorescence in each group of IEC-6 cells. Mitochondria was showed by white arrows in the pictures. The red fluorescence is indicative of the presence of ferrous ions. (E) The content of Fe2+ in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (F) The content of GSH in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (G) The content of ROS in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (H) The content of MDA in each group of IEC-6 cells and Caco-2 cells (n = 6 in each group). (I-K) IEC-6 cells were incubated 2 μM ferrostatin-1 (Fer-1) for 16 h, and then incubated with or without 10 ng/mL LPS for 12 h, followed by 200 μM DCA for 2 h. They were divided into six group (n = 5 or 6 in each group): control group, LPS group, LPS + DCA group, Fer-1 group, LPS + Fer-1 group and LPS + DCA + Fer-1 group. (I) The relative mRNA expression level of GPX4, ACSL4 and DMT1 in each group of IEC-6 cells (n = 6 in each group). (J) The content of GSH in each group of IEC-6 cells (n = 5 in each group). (K) The content of MDA in each group of IEC-6 cells (n = 5 in each group). Data are represented as mean ± SEM. ns P > 0.05, ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001, ∗∗∗∗P < 0.0001. ##P < 0.01, ####P < 0.0001. ∗ LPS group vs control group, LPS + DCA group vs LPS group. # LPS + Fer-1 group vs LPS group, LPS + DCA + Fer-1 group vs LPS + DCA group.

Figure 3 EPO regulates the expression of ferroptotic biomarkers after SCI. (A) Western blot of the indicated proteins in injured tissues from the treatment groups. (B–H) Quantitative analysis of Tfr, Fpn, Fth, Acsl4 and 4-Hne protein expression at 14 dpi. Gapdh was used as the reference protein. (I, J) Quantitative reverse transcription polymerase chain reaction results of xCT and Gpx4 mRNA extracted from injured tissue at 7 dpi. β-Actin mRNA was used as the reference gene. (K) Quantitative analysis of reduced GSH from injured tissue at 7 dpi. All data are expressed as the mean ± SD (n = 5 in each group). *P < 0.05, **P < 0.01, ***P < 0.001 (one-way analysis of variance followed by Dunnett’s multiple comparisons test). 4-Hne: 4-Hydroxynonenal; Acsl4: acyl-coenzyme A synthetase long chain family member 4; dpi: day(s) post injury; EPO: erythropoietin; Fpn: ferroportin or solute carrier family 40 member 1; Fth: ferritin heavy chain; Gapdh: glyceraldehyde-3-phosphate dehydrogenase; Gpx4: glutathione peroxidase 4; GSH: glutathione; ns: not significant; SCI: spinal cord injury; Tfr: transferrin receptor; xCT: the solute carrier family 7 member 11.