NCOA4 Antibody - #DF4255

Fig. 5 3-MA and NCOA4 knockdown suppress PS-NPs-induced ferritinophagy. (A) GO analysis based on proteomic analysis results of PS-NPs-treated group and control group. (B and C) Heat maps of ferroptosis-related and autophagy-related proteins in vehicle and PS-NPs-treated GC-2 cells. (D) Immunofluorescence of LC3 in GC-2 cells treated with PS-NPs. (E) Relative expression of ferritinophagy-related gene in GC-2 cells post-treatment PS-NPs. (F and G) Ferritinophagy-related protein expression in PS-NPs-stimulated GC-2 cells. (H) Immunofluorescence of NCOA4 and FTH1 in GC-2 cells treated with PS-NPs. Line intensity plots show colocalization between FTH1 (red) and NCOA4 (green). (I) The cell viability was assayed in GC-2 cells following PS-NPs treatment with or without 3-MA. (J) Immunofluorescence staining of LC3 in GC-2 cells post-treatment with PS-NPs and 3-MA. (K and L) Western blotting and quantitative analysis of NCOA4 and FTH1. (M) Immunofluorescence of NCOA4 and FTH1 in GC-2 cells following PS-NPs and 3-MA treatment. Line intensity plots show colocalization between FTH1 (red) and NCOA4 (green). (N) The viability of PS-NPs-stimulated GC-2 cells while simultaneously inhibiting NCOA4 expression. (O and P) Representative fluorescent images and quantitative analysis of lipid peroxidation levels in PS-NPs-stimulated GC-2 cells while simultaneously inhibiting NCOA4 expression. (Q and R) Intracellular chelatable iron in GC-2 cells after 12 h exposure to PS-NPs while inhibiting NCOA4 expression stained with FerroOrange. (S and T) Representative fluorescent images and quantification of ROS levels using the fluorescent indicator DCFH-DA in GC-2 cells after 12 h exposure to PS-NPs while inhibiting NCOA4 expression

Fig. 5 3-MA and NCOA4 knockdown suppress PS-NPs-induced ferritinophagy. (A) GO analysis based on proteomic analysis results of PS-NPs-treated group and control group. (B and C) Heat maps of ferroptosis-related and autophagy-related proteins in vehicle and PS-NPs-treated GC-2 cells. (D) Immunofluorescence of LC3 in GC-2 cells treated with PS-NPs. (E) Relative expression of ferritinophagy-related gene in GC-2 cells post-treatment PS-NPs. (F and G) Ferritinophagy-related protein expression in PS-NPs-stimulated GC-2 cells. (H) Immunofluorescence of NCOA4 and FTH1 in GC-2 cells treated with PS-NPs. Line intensity plots show colocalization between FTH1 (red) and NCOA4 (green). (I) The cell viability was assayed in GC-2 cells following PS-NPs treatment with or without 3-MA. (J) Immunofluorescence staining of LC3 in GC-2 cells post-treatment with PS-NPs and 3-MA. (K and L) Western blotting and quantitative analysis of NCOA4 and FTH1. (M) Immunofluorescence of NCOA4 and FTH1 in GC-2 cells following PS-NPs and 3-MA treatment. Line intensity plots show colocalization between FTH1 (red) and NCOA4 (green). (N) The viability of PS-NPs-stimulated GC-2 cells while simultaneously inhibiting NCOA4 expression. (O and P) Representative fluorescent images and quantitative analysis of lipid peroxidation levels in PS-NPs-stimulated GC-2 cells while simultaneously inhibiting NCOA4 expression. (Q and R) Intracellular chelatable iron in GC-2 cells after 12 h exposure to PS-NPs while inhibiting NCOA4 expression stained with FerroOrange. (S and T) Representative fluorescent images and quantification of ROS levels using the fluorescent indicator DCFH-DA in GC-2 cells after 12 h exposure to PS-NPs while inhibiting NCOA4 expression

Fig. 6 Ferroptosis was triggered by MtROS-dependent autophagy. (A) Effects of NaAsO2 (4  and TEMPO on LC3 and p62 in MIN6 cells. (B) Autophagic flux analysis. (C) The efficiency of CQ, and its effect on GPX4 and COX-2 in NaAsO2- Journal Pre-proof treated MIN6 cells. (D) Effects of NaAsO2 and CQ on relative GSH, T-SOD, and MDA content in MIN6 cells. (E) Effects of CQ on the NaAsO2-induced accumulation of lipid ROS by BODIPY 581/591 C11 staining (scale bar = 50 m). (F) Effects of CQ on the NaAsO2-induced insulin released using an ELISA kit. All results are expressed as the mean ± SD, n = 3. *P < 0.05, **P < 0.001 vs. control group, #P < 0.05, ##P < 0.001 vs. NaAsO2 group.

Fig. 8 Autophagy triggered ferroptosis in CuSO4-treated GC-1 spg cells. (a) Cells were treated with CuSO4 (0, 0.4, 0.8 and 1.6 mM) for 12h and 24h. The Western blot results of GPX4 and COX-2 protein expression. (b) The quantification of GPX4 and COX-2. (c) Cells were treated with CuSO4 (1.6 mM, 24h) in the presence/absence of 3-MA (1 mM, 1h), the changes of GPX4, COX-2, NCOA4 and FTH1. (d) Cells transfected with control shRNA and ATG5 shRNA exposed with CuSO4 (1.6 mM) for 24 h, changes of GPX4, COX-2, NCOA4 and FTH1. Data are presented with the means ± standard deviation. *p < 0.05 **p < 0.01.

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