Journal: Molecular and cellular biochemistry

Article Title: Soluble nickel interferes with cellular iron homeostasis.

doi: 10.1007/s11010-005-8288-y

Figure Lengend Snippet: Fig. 2. Effects of nickel on ferritin induction by TfR bound iron and NTBI. (a) A549 cells were treated with 1 mM NiCl2, 500 µM FeSO4, 500 µM FeSO4 + 500 µM NiCl2, 500 µM FeSO4 + 1 mM NiCl2 for 24 h. (b) A549 cells were treated with 1 mM NiCl2, 25 µg/ml Holo-transferrin, 25 µg/ml Holo-transferrin+500 µMNiCl2,25 µg/mlHolo-transferrin+1 mMNiCl2 for 24 h. Cellular protein extracts were isolated as described in the section on ‘Materials and methods.’ Thirty micrograms of protein were subjected to western blot analysis and primary antibodies were used to determine the presence of ferritin light chain. Antibodies for α-tubulin were used to confirm equal loading of proteins.

Article Snippet: Immunoblotting was performed with diluted ferritin light chain antibody (1:300) (Santa Cruz Biotechnology, Santa Cruz, CA).

Techniques: Isolation, Western Blot

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: The uremic toxin indoxyl sulfate interferes with iron metabolism by regulating hepcidin in chronic kidney disease.

doi: 10.1093/ndt/gfx252

Figure Lengend Snippet: FIGURE 4: Changes in the expression of duodenal iron metabolism–related proteins in mice. Upper panel: Representative immunoblots for (A) FPN, (B) DMT1, (C) FTH and (D) FTL and protein staining. Lower panel: Semiquantitative densitometric analyses of FPN, DMT1, FTH and FTL protein levels normalized to protein staining. Values are expressed as mean 6 SD. *P < 0.05; n ¼ 11–15 in each group.

Article Snippet: The following commercially available antibodies were used for this study: anti-NRAMP2 (DMT1) antibody, anti-ferritin heavy chain (FTH), anti-ferritin light chain (FTL) and anti-AhR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); antitransferrin receptor 1 (TfR1) antibody (Zymed Technologies, Carlsbad, CA, USA); anti-FPN antibody (Alpha Diagnostics, San Antonio, TX, USA); anti-a-tubulin (Merck KGaA, Darmstadt, Germany), which was used as a protein loading control and antihistone H3 antibody (Abcam, Cambridge, UK), which was used as a loading control in the case of nuclear proteins.

Techniques: Expressing, Western Blot, Staining

Journal: Nephrology, dialysis, transplantation : official publication of the European Dialysis and Transplant Association - European Renal Association

Article Title: The uremic toxin indoxyl sulfate interferes with iron metabolism by regulating hepcidin in chronic kidney disease.

doi: 10.1093/ndt/gfx252

Figure Lengend Snippet: FIGURE 5: Alterations in iron metabolism–related proteins in the spleen of mice. Upper panel: Representative immunoblots for (A) FPN, (B) DMT1, (C) TfR, (D) FTH and (E) FTL and tubulin. Lower panel: Semiquantitative densitometric analyses of FPN, DMT1, TfR, FTH and FTL protein levels normalized to tubulin. Values are expressed as mean 6 SD. *P < 0.05, **P < 0.01; n ¼ 14–18 in each group. The mRNA expres- sion of (F) hepcidin and (G) FPN in the spleen of mice. Values are expressed as mean 6 SD. *P < 0.05, **P < 0.01; n ¼ 7–12 in each group. (H) Upper panel: Representative IRP–IRE-binding bands. Lower panel: Semiquantitative densitometry analysis of IRP activity in the spleen. *P < 0.05, **P < 0.01; n ¼ 5 in each group.

Article Snippet: The following commercially available antibodies were used for this study: anti-NRAMP2 (DMT1) antibody, anti-ferritin heavy chain (FTH), anti-ferritin light chain (FTL) and anti-AhR antibody (Santa Cruz Biotechnology, Santa Cruz, CA, USA); antitransferrin receptor 1 (TfR1) antibody (Zymed Technologies, Carlsbad, CA, USA); anti-FPN antibody (Alpha Diagnostics, San Antonio, TX, USA); anti-a-tubulin (Merck KGaA, Darmstadt, Germany), which was used as a protein loading control and antihistone H3 antibody (Abcam, Cambridge, UK), which was used as a loading control in the case of nuclear proteins.

Techniques: Western Blot, Binding Assay, Activity Assay

Journal: Redox biology

Article Title: Estradiol-mediated small GTP-binding protein GDP dissociation stimulator induction contributes to sex differences in resilience to ferroptosis in takotsubo syndrome.

doi: 10.1016/j.redox.2023.102961

Figure Lengend Snippet: Fig. 3. SmgGDS knockdown exacerbates ferritinophagy-mediated ferroptosis in female mice. A, The mRNA level of PTGS2 in cardiac tissues of each group. n = 4, each. B, Representative images of immunohistochemistry staining for PTGS2 and FTH1 protein in cardiac tissues of each group and quantification. Scale bars, 20 μm. C, Representative images of Prussian blue staining for iron deposition in the hearts of each group and quantification. n = 4, each. Scale bars, 50 μm. D, The cardiac MDA level in female WT and SmgGDS ± mice after 2 h of saline or ISO injection. n = 3, each. E, Western blot analysis of PTGS2, P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I expression in female WT and SmgGDS ± mice after saline or ISO treatmen. n = 4, each. ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing saline vs ISO treatment. (For interpretation of the references to color in this figure legend, the reader is referred to the Web version of this article.)

Article Snippet: For immunohistochemistry, the heart sections were incubated with primary antibodies, including CD45 (Servicebio, GB11066, 1:1000), MCP-1 (Servicebio, GB11199, 1:500), PTGS2 (ABclonal, A1253, 1:50) and FTH1 (ABclonal, A1144, 1:50) overnight at 4 ◦C, and then followed by incubation with the immunohistochemistry commercial kit (ZSGB-Bio, PV-9001).

Techniques: Knockdown, Immunohistochemistry, Staining, Saline, Injection, Western Blot, Expressing

Journal: Redox biology

Article Title: Estradiol-mediated small GTP-binding protein GDP dissociation stimulator induction contributes to sex differences in resilience to ferroptosis in takotsubo syndrome.

doi: 10.1016/j.redox.2023.102961

Figure Lengend Snippet: Fig. 5. SmgGDS modulates E2-mediated anti-ferritinophagy in ISO-induced HL-1 cardiomyocytes. A, MDA in sh-NC and sh-SmgGDS HL-1 cardiomyocytes treated with PBS or ISO (10 mM) for 2 h n = 3, each. B, Western blot analysis of P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I in sh-NC and sh-SmgGDS HL-1 cardiomyocytes after treatment with PBS or ISO (10 mM) for 2 h n = 3, each. C, Representative images of FerroOrange staining for intracellular Fe2+ in HL-1 cardiomyocytes of each group and quantification. n = 4, each. D, Representative images of intracellular lipid ROS by BODIPY probe in PBS or ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without Fer-1 (10 μM) or 3-MA (5 μM); the fluorescence intensity in each group was quantified by ImageJ. n = 4, each. E, Representative images of DCFH-DA staining in PBS or ISO-challenged HL-1 cardiomyocytes pretreated with or without glutathione S- transferase (GST)-tagged recombinant SmgGDS protein (GST-SmgGDS, 1 μg/mL); the fluorescence intensity in each group was quantified by ImageJ. n = 4, each. F, Western blot analysis of PTGS2, SLC7A11, GPX4, FTH1, P62 and LC3B-II/LC3B–I expression in HL-1 cardiomyocytes pretreated with or without GST-SmgGDS (1 μg/ mL) for 24 h followed by ISO stimulation. n = 3, each. G, Western blot analysis of SmgGDS, PTGS2, P62, SLC7A11, FTH1, GPX4 and LC3B-II/LC3B–I expression in ISO-challenged sh-NC and sh-SmgGDS HL-1 cardiomyocytes pretreated with or without E2. n = 4, each. ns, P ≥0.05; #P < 0.05, ##P < 0.01, ###P < 0.001; *P < 0.05, **P < 0.01, ***P < 0.001 comparing PBS vs ISO treatment.

Article Snippet: For immunohistochemistry, the heart sections were incubated with primary antibodies, including CD45 (Servicebio, GB11066, 1:1000), MCP-1 (Servicebio, GB11199, 1:500), PTGS2 (ABclonal, A1253, 1:50) and FTH1 (ABclonal, A1144, 1:50) overnight at 4 ◦C, and then followed by incubation with the immunohistochemistry commercial kit (ZSGB-Bio, PV-9001).

Techniques: Western Blot, Staining, Fluorescence, Recombinant, Expressing

Journal: Journal of clinical biochemistry and nutrition

Article Title: Intracellular ferritin heavy chain plays the key role in artesunate-induced ferroptosis in ovarian serous carcinoma cells.

doi: 10.3164/jcbn.21-82

Figure Lengend Snippet: Fig. 4. Effect of FTH gene modulation on ART-induced cell death. (A) Profile of FTH level by Western blotting analysis in CaOV3 and SKOV3ip1 cells and FTH silenced in SKOV3ip1 cells by siRNA transfection. (B) Effect of FTH silencing on ART-induced reduction of cell viability in CaOV3 and SKOV3ip1 cells after 24 h of treatment. (C, D) Effect of FTH silencing on ART (70 μM)-enhanced lipid peroxidation in SKOV3ip1 cells after 24 h of treatment. C represents profile of BODIPY 581/591 C11. n = 7. (E, F) Effect of FTH silencing on ART (70 μM)-induced intracellular Fe2+ in SKOV3ip1 cells after 5 h of treatment. E represents profile of Lyso-RhoNox. n = 7. (G) Western blotting analysis showed that FTH was overexpressed in CaOV3 cells through cDNA plasmid transfection. (H) Effect of FTH overexpression on ART-induced reduction of cell viability in CaOV3 after 24 h of treatment. n = 5. (I, J) Effect of FTH overexpression on ART (70 μM)-enhanced lipid peroxidation in CaOV3 cells after 24 h of treatment. I represents profile of BODIPY 581/591 C11. n = 7. (K, L) Effect of FTH overexpression on ART (70 μM)-induced intracellular Fe2+ in CaOV3 cells after 5 h of treat‐ ment. K represents profile of Lyso-RhoNox. n = 7. DMSO was used as vehicle and scrambled siRNA and empty vector (EV) were used as control. FTH cDNA represents FTH overexpression. *p<0.05.

Article Snippet: FTH1 cDNA plasmid (RC209845; OriGene, Rockville, MD) was used for gene overexpression. pCMV6-Entry Mammalian Expression Vector [empty vector (EV), PS100001; OriGene] were used for negative control.

Techniques: Western Blot, Transfection, Plasmid Preparation, Over Expression, Control