FTH1 Antibody - #DF6278

Fig. 5. UIRI induces ferroptosis in renal tubular epithelial cells. A. Diagram shows the animal experimental design. B, C. Serum creatinine (B) and blood urea nitrogen (C) levels in different groups as indicated (n = 3). D. Representative images of HE, Perl's staining, and immunohistochemical staining for SLC7A11 and GPX4 in UIRI or Fer-1 (5 mg/kg) treated mice kidneys. Scale bar = 50um. E. mRNA expression of SLC7A11, FTH-1, and GPX4 in UIRI or Fer-1 treated mice kidneys was measured by qRT-PCR (n = 3). F. Representative Western blotting (Fi) and quantitative data (Fii) show the protein expression of SLC7A11, FTH-1, and GPX4 in Fer-1 treated mice kidneys after UIRI. Relative expressions were expressed as fold induction over sham controls after normalization with GAPDH. ns, ***P < 0.001 and ****P < 0.0001 vs sham; ####P < 0.0001 vs UIRI group; ns, not significant (n = 4). G-I. MDA (G), GSSG/total GSH ratio (H), and Iron content (I) in different treated kidney tissues (n = 4, ***P < 0.001 and ****P < 0.0001 versus sham; ##P < 0.01 and ###P < 0.001 vs UIRI group). J. Double immunofluorescence staining demonstrates ferroptosis predominantly in the proximal tubular epithelium. Kidney sections were co-stained for AQP-1 (Red) and GPX4 (green), respectively. Scale bar = 50 μm.

Figure 2 VDR activation suppressed ferroptosis in HG‐cultured HK‐2 cells. The mitochondrial morphology of HK‐2 cells was visualized by TEM. The arrow indicates microscopic changes in mitochondria. Absolute counting of the number of cristae per mitochondria. Scale bar = 2 or 0.5 µm (A). Quantitative analysis of iron (B), MDA (C), and GSH (D) levels in each cell group. The relative mRNA levels of SLC7A11, GPX4, TFRC,F TH1, and AIFM2 in different groups of HK‐2 cells were determined by qRT‐PCR. ACTB served as a loading control (E). Western blotting was applied to detect the protein expression levels of SLC7A11, GPX4, TFR‐1, FTH‐1, and FSP1, followed by densitometric analysis of the blots. β‐actin served as a loading control (F). Immunofluorescence staining and fluorescence intensity analysis of SLC7A11, GPX4, and TFR‐1 in different groups of HK‐2 cells as indicated. Scale bar = 20 µm (G). Each bar represents the mean ± SD of the data derived from three independent experiments (n = 3). ** p < 0.01 versus NG group; # p < 0.05, ## p < 0.01 versus HG group; & p < 0.05, && p < 0.01 versus HG group.

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. 5. Disordered iron homeostasis and inhibited mTORC1 activity upon exposure to CdCl2 and PCB77 at low dose. (A) The relative fluorescence intensity of CAeAM for measuring LIP to reflect intracellular iron availability (n = 3–4), and (B) Representative blots of FTH1 protein content to reflect iron storage. Analyses were performed after single or combined exposure to CdCl2 and PCB77 at 1 μM for 48 h. (C) Phosphorylated S6 and total S6 content to re- flect mTORC1 activity as measured by Western blot. Ratio of FTH1 to eIF2αP and ratio of pS6 to S6 in the control group were defined as 1. Analyses were performed after single or combined exposure to CdCl2 and PCB77 at 1 μM for 48 h. a- significantly different from the control group. Data were presented in mean ± SE. P < 0.05 was considered statistically significant.

Fig. 5 ZIP8 regulates ferroptosis of monocytes under sepsis conditions in vitro. a. Representative immunoblots of ZIP8-G, ZIP8-N, GPX4, FTH1 and xCT in RAW264.7 cells treated with different concentrations of LPS and durations. b-f. Immunoblot analyses of ZIP8-G (b, n = 4/group), ZIP8-N (c, n = 4/group), GPX4 (d, n = 4/group), FTH1 (e, n = 4/group), and xCT (f, n = 4/group) exhibited a sequential pattern of expression changes between ZIP8 (peaked at 6 h post-treatment) and ferroptosis related GPX4 and FTH1 (hit the lowest at 12 h post-treatment). g. Representative immunoblots of ZIP8-G, ZIP8-N, GPX4, FTH1 and xCT in RAW264.7 cells with/without decreased Slc39a8 expression and/or LPS treatment. h-l. Immunoblot analyses of ZIP8-G (h, n = 5/group), ZIP8-N (i, n = 5/group), GPX4 (j, n = 5/group), FTH1 (k, n = 5/group), and xCT (l, n = 5/group) exhibited that suppressing ZIP8 attenuated the decrease of GPX4, FTH1 and xCT in response to LPS. m-o. Quantification analysis showed that decreasing ZIP8 alleviated the LPS-induced upregulations of MDA (m, n = 6/group) and ferrous ions (o, n = 6/group) and reversed the decreased GSH level (n, n = 6/group). One-way ANOVA was performed in b-f. Two-way ANOVA was performed in h-o. Compared to PBS treated si-NC group:

Fig. 5. FTH1 plays a critical role in silibinin-mediated inhibition of ferroptosis, and silibinin inhibits ferroptosis by disrupting the NCOA4-FTH1 interaction. (A) The schematic diagram of identifying silibinin target proteins. (B) Venn diagram of silibinin target proteins and biotin target proteins. (C) Representative images of FTH1 in the proteome microarray. (D) Z-score and IMean ratio of FTH1-silibinin binding. (E) Molecular docking of FTH1 and silibinin. (F) SPRi fitting curves for silibinin to FTH1. (G–H) CETSA-Western blot analysis showed the protection of FTH1 by silibinin at different temperature gradients (n = 3). (I–J) DARTS-Western blot analysis showed the resistance of FTH1 to pronase digestion under the treatment of silibinin (n = 3). (K–M) Western bolt analysis and quantitative results of NCOA4 and FTH1 (n = 4). (N) Representative immunofluorescence images of FTH1 and NCOA4 co-staining. (O) Co-IP assay of NCOA4 and FTH1 interaction in the kidney of IRI mice. (P) Quantitative results of co-IP assay showing the endogenous interaction between NCOA4 and FTH1 (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

Fig. 5. FTH1 plays a critical role in silibinin-mediated inhibition of ferroptosis, and silibinin inhibits ferroptosis by disrupting the NCOA4-FTH1 interaction. (A) The schematic diagram of identifying silibinin target proteins. (B) Venn diagram of silibinin target proteins and biotin target proteins. (C) Representative images of FTH1 in the proteome microarray. (D) Z-score and IMean ratio of FTH1-silibinin binding. (E) Molecular docking of FTH1 and silibinin. (F) SPRi fitting curves for silibinin to FTH1. (G–H) CETSA-Western blot analysis showed the protection of FTH1 by silibinin at different temperature gradients (n = 3). (I–J) DARTS-Western blot analysis showed the resistance of FTH1 to pronase digestion under the treatment of silibinin (n = 3). (K–M) Western bolt analysis and quantitative results of NCOA4 and FTH1 (n = 4). (N) Representative immunofluorescence images of FTH1 and NCOA4 co-staining. (O) Co-IP assay of NCOA4 and FTH1 interaction in the kidney of IRI mice. (P) Quantitative results of co-IP assay showing the endogenous interaction between NCOA4 and FTH1 (n = 3). ∗P < 0.05, ∗∗P < 0.01, ∗∗∗P < 0.001.

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 2. Elevation of xanthurenic acid level aggravates myocardial ischemia injury. A. Serum biochemical indicators contents include BNP, hs-CRP, TNF-α, MDA, and NO in sham and MI model groups mice administered xanthurenic acid (XA), and were detected by ELISA (i.p. 100 mg/kg) (n = 6). B-D. Histological analysis of heart, lung, liver, spleen, kidney, and brain slices by hematoxylin and eosin (H&E), masson's trichrome, and sirius red staining (n = 5) (All images were 200X magnification except for spleen was 400X magnification; the lines marked in all figures represent 50 µm). E. Western blots and quantitative results of cleaved caspase-3, GPX4, FHC, FLC, and ACSL4 in each group mice heart (n = 5). Data are presented as mean ± SD. #p < 0.05, ##p < 0.01 vs. sham group, *p < 0.05, **p < 0.01 vs. model group.