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

Journal: Military Medical Research

Article Title: Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease.

doi: 10.1186/s40779-023-00487-3

Figure Lengend Snippet: Fig. 5 Cav-1 deficiency in hepatocytes aggravated the accumulation of liver Fe2+ in NAFLD. a Schematic construction of hepatocellular-specific Cav-1 knockdown mice. b HE staining and Nile Red staining results of liver tissue. c The concentration of serum Cav-1 between Flox mice and Cav-1-CKO mice with or without high-fat diet (HFD) (n = 4). d The concentration of serum total iron, serum Tf, serum hepcidin, and serum ferritin in different groups (n = 4). e The concentration of Fe2+ and Fe3+ in liver tissue between Flox mice and Cav-1-CKO mice with or without HFD (n = 4). f Prussian blue staining results in different groups. g The expression of FTL in liver tissue was detected by Western blotting (n = 3). h Immunohistochemical method detected the expression of FTL and FTH protein in liver tissue (n = 4). i The expression of Fpn1 and hepcidin in liver tissue between Flox mice and Cav-1-CKO mice with HFD (n ≥ 3). *P < 0.05, **P < 0.01, ***P < 0.001, as determined by Student’s t test analysis or one-way ANOVA. All data were shown as the mean ± SD. Cav-1-CKO Cav-1-knockout mice, Cav-1 caveolin-1, NAFLD non-alcoholic fatty liver disease, Tf transferrin, FTL ferritin light chain, FTH ferritin heavy chain, Fpn1 ferroportin 1, PA palmitic acid, OA oleic acid

Article Snippet: ECL detection reagents were used to detect the expression of related proteins (Millipore, USA), for example: Cav-1 (rabbit, #3238, CST, USA), GAPDH (rabbit, #8884, Abcam, UK), β-actin (mouse, 60008-1-Ig, Proteintech, China), HO-1 (rabbit, ab13243, Abcam, UK), nuclear factor erythroid 2-related factor 2 (Nrf2; rabbit, 16396-1-AP, Proteintech, China), Fpn1 (rabbit, NBP1-21502, NOVUS, USA), FTL (rabbit, 10727-1-AP, Proteintech, China), FTH (rabbit, A19544, ABclonal, China), and fatty acid synthase (FASN; rabbit, ab128870, Abcam).

Techniques: Knockdown, Staining, Concentration Assay, Expressing, Western Blot, Immunohistochemical staining, Knock-Out

Journal: Military Medical Research

Article Title: Caveolin-1 is critical for hepatic iron storage capacity in the development of nonalcoholic fatty liver disease.

doi: 10.1186/s40779-023-00487-3

Figure Lengend Snippet: Fig. 8 Scheme of Cav-1 inhibiting the development of NAFLD by regulating iron metabolism. The HFD inhibited the expression of Cav-1 in hepatocytes and up-regulated the expression of Cav-1 in liver macrophages. In hepatocytes, Cav-1 regulated the FTL/FTH signaling pathway to promote the conversion of Fe2+ to Fe3+, which helped to slow down the oxidative stress induced by Fe2+ and ultimately alleviate the progression of NAFLD. In macrophages, Cav-1 regulated the expression of HO-1 in CD68+CD163+ macrophages, and promoted the degradation of red blood cells into Fe2+ within macrophages which ultimately exacerbated the disorder of iron metabolism in the liver. In addition, the concentration of ferritin and transferrin in serum was up-regulated by Cav-1, and the two were positively correlated in human subject. HFD high-fat diet, Cav-1 caveolin-1, Tf transferrin, NAFLD non-alcoholic fatty liver disease, HO-1 heme oxygenase-1, FTL ferritin light chain, FTH ferritin heavy chain, RBC red blood cell, ROS reactive oxygen species

Article Snippet: ECL detection reagents were used to detect the expression of related proteins (Millipore, USA), for example: Cav-1 (rabbit, #3238, CST, USA), GAPDH (rabbit, #8884, Abcam, UK), β-actin (mouse, 60008-1-Ig, Proteintech, China), HO-1 (rabbit, ab13243, Abcam, UK), nuclear factor erythroid 2-related factor 2 (Nrf2; rabbit, 16396-1-AP, Proteintech, China), Fpn1 (rabbit, NBP1-21502, NOVUS, USA), FTL (rabbit, 10727-1-AP, Proteintech, China), FTH (rabbit, A19544, ABclonal, China), and fatty acid synthase (FASN; rabbit, ab128870, Abcam).

Techniques: Expressing, Concentration Assay

Journal: eLife

Article Title: cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool

doi: 10.7554/eLife.29750

Figure Lengend Snippet: ( A ) cAMP (100 μM) treatment induces labile Fe(II) detected by Trx-Puro probes at a comparable level in Ferritin heavy chain knockdown cells compared to the cells treated with scramble siRNA or with no pretreatment. Scale bar = 20 μm. ( B ) IF quantification indicates the knocking down Ferritin does not abolish labile Fe(II) induced by cAMP. ( C ) Immunoblot of Ferritin shows that the level of Ferritin heavy chain and Ferritin complex are decreased by siRNA compared to cells transfected by scramble siRNA. p<0.0005 (n = 2 independent experiments with three biological replicates for IF and two biological replicates for immunoblot).

Article Snippet: To decrease the expression of Ferritin, a pool of three siRNA duplexes (cat# SR301663, Origene Technologies, Rockville, MD) against FTH1 and scramble siRNA (cat# SR30004) were used.

Techniques: Knockdown, Western Blot, Transfection

Journal: eLife

Article Title: cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool

doi: 10.7554/eLife.29750

Figure Lengend Snippet: ( A ) Pretreatment with PKA inhibitors (KT5720 (2 μM), H89 (20 μM)), Epac inhibitor ESI09 (10 μM) or CNGC blocker LCD (10 μM) for 20 min prior to cAMP addition showed no effect on labile Fe(II) induced by cAMP (100 μM) treatment in Schwann cells detected by Trx-Puro probes. ( B ) Knocking down the expression of RapGEF2 in HEK-293 cells largely blocked the acidification of vesicles and labile Fe(II) elevation after cAMP (100 μM) treatment compared to the scramble siRNA group. ( C ) IF quantification indicates that knocking down RapGEF2 inhibits but does not completely abolish vesicle acidification induced by cAMP. ( D ) IF quantification indicates that knocking down RapGEF2 inhibits but does not completely abolish labile Fe(II) induced by cAMP. Scale bar = 20 μm. p<0.0005 (n = 3 independent experiments with three biological replicates in each group). 10.7554/eLife.29750.022 Figure 5—source data 1. Fragments per kilobase per million (FPKM) of CNG and Rapgef genes in Schwann cells.

Article Snippet: To decrease the expression of Ferritin, a pool of three siRNA duplexes (cat# SR301663, Origene Technologies, Rockville, MD) against FTH1 and scramble siRNA (cat# SR30004) were used.

Techniques: Expressing

Journal: eLife

Article Title: cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool

doi: 10.7554/eLife.29750

Figure Lengend Snippet: qRT-PCR shows that the mRNA level of RapGEF2 is lower in the siRNA group compared to the scramble siRNA group (p=0.042) (n = 3 independent experiments with three biological replicates each, error bars denote standard error).

Article Snippet: To decrease the expression of Ferritin, a pool of three siRNA duplexes (cat# SR301663, Origene Technologies, Rockville, MD) against FTH1 and scramble siRNA (cat# SR30004) were used.

Techniques: Quantitative RT-PCR

Journal: eLife

Article Title: cAMP signaling regulates DNA hydroxymethylation by augmenting the intracellular labile ferrous iron pool

doi: 10.7554/eLife.29750

Figure Lengend Snippet: ( A ) Knocking down the expression of Rap1 largely blocked the effect of cAMP (100 μM) on vesicle acidification and labile Fe(II) elevation detected by Trx-Puro probes, while knocking down RAP2 and scramble siRNA had no obvious effect. Scale bar = 20 μm. ( B ) IF quantification indicates the inhibition of RAP1 siRNA on cAMP-induced vesicle acidification. ( C ) IF quantification indicates the inhibition of RAP1 siRNA on cAMP-induced labile Fe(II). ( E ) qRT-PCR shows the mRNA level of Rap isoforms are lower in the siRNA group compared to the scramble siRNA group. *p<0.005 (n = 1 independent experiment with three biological replicates each, error bars denote standard error).

Article Snippet: To decrease the expression of Ferritin, a pool of three siRNA duplexes (cat# SR301663, Origene Technologies, Rockville, MD) against FTH1 and scramble siRNA (cat# SR30004) were used.

Techniques: Expressing, Inhibition, Quantitative RT-PCR

Journal: Molecular metabolism

Article Title: Adipose knockout of H-ferritin improves energy metabolism in mice.

doi: 10.1016/j.molmet.2024.101871

Figure Lengend Snippet: Figure 4: Fth1 deletion in the adipose tissue caused upregulation of mitochondrial biogenesis and respiratory capacity and a tendency of iWAT browning. (A,B) qRT- PCR analysis of Pgc1a, Nrf1, Ndufs1, Sdhc, Cox4, Cox8b, and Atp5a1 expression in iBAT and iWAT of Fth AKO and Fthfl/flmice (n ¼ 4e6, from two independent experiments). (C) NADH consumption assay of mitochondria isolated from Fth/ and Fthfl/flMEFs (n ¼ 5, from 3 independent experiments). (D) The activities of mitochondrial ETC complex I and II of mitochondria isolated from mature adipocytes of iBAT and iWAT (n ¼ 3e5, from two independent experiments). (E,F) qRT-PCR analysis of Ucp1, Car4, Dio2, Elovl3, Cidea, and Serca2 expression in iBAT and iWAT of Fth AKO and Fthfl/flmice (E: n ¼ 4e6; F: n ¼ 3e6, from three independent experiments). (G) Rectal temperature of Fth AKO and Fthfl/flmice housed at room temperature or 4 C (n ¼ 6, from three independent experiments). (H) Free fatty acid in mice serum before and after cold exposure test (n ¼ 4e5, from two independent experiments). (I) Serum Il-6 of Fth AKO and Fthfl/flmice housed at 4 C (n ¼ 3). Statistical significance of the difference between Fth/ and Fthfl/flin Figure 4C was determined by two-way ANOVA followed by Sidak’s multiple comparisons. Other data was analyzed by two-tailed unpaired t-tests. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, and ns: not significant (p > 0.05).

Article Snippet: Animals Mice referred to as FthAKO mice are conditional adipocyte Fth1 knockout (Adipoq-Cre; Fthfl/fl) mice, generated by intercrossing Fthfl/fl mice (Jackson Laboratory, stock #018063) [28] with mice carrying the adiponectin promoter-cre transgene (Cyagen Biosciences, Suzhou, China) [29].

Techniques: Quantitative RT-PCR, Expressing, Isolation, Two Tailed Test