GPX4 Antibody - #DF6701

Fig. 6 ZnO NPs induce the ferroptosis of GC-2 cells. (A) Intracellular chelatable iron in GC-2 cells treated with or without ZnO NPs stained with PGSK (green). Statistical analysis of MFI of PGSK was shown. (B) Representative FACS data for lipid peroxidation level in GC-2 cells following ZnO NPs treatment using C11 BODIPY. Statistical analysis of MFI of the ratio of green/red was shown. (C) qRT-PCR analysis of ferroptosis-related gene expression in GC-2 cells after ZnO NPs treatment. (D) Western blot of ferroptosis-related protein levels in GC-2 cells treated with ZnO NPs. Statistical analysis of mean grey values ratios of the corresponding proteins/β-actin was shown, the same as below. (E) The cell viability of GC-2 cells following ZnO NPs treatment with or without Fer-1 (3.5 µM). (F) Intracellular chelatable iron in GC-2 cells following ZnO NPs treatment with or without Fer-1 stained with PGSK. Statistical analysis of MFI of PGSK was shown. (G) Representative FACS data of lipid peroxidation level in GC-2 cells following ZnO NPs treatment with or without Fer-1 stained with C11 BODIPY. Statistical analysis of MFI of the ratio of green/red was shown. (H and I) The levels of GSH and MDA in GC-2 cells following ZnO NPs treatment with or without Fer-1. (J) qRT-PCR analysis of ferroptosis-related gene expression in GC-2 cells following ZnO NPs treatment with or without Fer-1. (K) Western blot of ferroptosis-related protein levels in GC-2 cells following ZnO NPs treatment with or without Fer-1

Figure 3. Synergistic tumor inhibition of CP and CQ dependent on ROS generation and irrespective of autophagosome accumulation. (A) Tumor volume curves were monitored during different treatments by intratumoral injection. CP, 5 mg/kg; CQ, 8.56 mg/kg; wortmannin (Wort), 0.35 mg/kg. n=5. (B and C) Photograph images and average tumor weights of the dissected tumors after the indicated treatments. n=5. (Dand E) Western blot analysis (D) of CT-26 cells exposed to the indicated treatments in the presence or absence of ATG5 expression attenuation. Image J software was used (E). CP, 15 μg/mL; CQ, 50 μM. Triple individual experiments were performed. (F) Cell viability analysis of CT-26 cells after the indicated treatments for 24 h in the presence or absence of ATG5 expression attenuation. CP, 15 μg/mL; CQ, 50 μM. Triple individual experiments were performed. n=3. (G and H) ROS analysis by flow cytometry after CT-26 after exposed to different treatments for 24 h and then stained with DCFH-DA (10 μM; 20 min). Triple individual experiments were performed. (I and J) Cell viability analysis (24 h treatment) and ROS analysis (6 h treatment) of CT-26 cells after the indicated treatments. CP, 15 μg/mL; CQ, 50 μM; NAC, 5 mM. (K) Total copper contents in CT-26 cells after the indicated treatments for 12 h was quantified by ICP-OES. CQ, 50 μM. (L) Fluorescence images stained by R6G (10 μΜ; 20 min) for CT-26 after the indicated treatments for 6 h. CP, 15 μg/mL; CQ, 50 μΜ. λex= 495 nm. (M) Western blot analysisof CT-26 cells after the indicated treatments for 24 h. CP, 15 μg/mL; CQ, 50 μM.

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.

Fig. 8. GPX4 inhibitor RSL3 substantially attenuates VK2 protection of chondrocytes. (A-I) WB results for GPX4, NFκB, pMAPK/MAPK, Aggrecan, CollagenⅡ, SOX9, MMP3, MMP13 and Tubulin; (J) Quantitive analysis of relative Glutathione Peroxidase Activity; (K-L) GSH contents and ratio of GSH/GSSG; (M) Quantitative results of MDA content assay.