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: 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

Journal: Journal of Cardiothoracic Surgery

Article Title: NSUN2 inhibits NCOA4 expression to alleviate ferroptosis and inflammation in sepsis-induced myocardial injury in a m 5 C manner

doi: 10.1186/s13019-025-03554-z

Figure Lengend Snippet: NSUN2 suppressed ferroptosis in LPS-induced SIMI. Cellular A , Fe 2 + level, B , GSH content, and C , relative ROS production in each group were determined by commercial kits; D , Western blot was performed to assess the protein levels of NCOA4, FTH1, and GPX4 in each group Fe 2+ , ferrous iron; GSH, glutathione; ROS, reactive oxygen species; NCOA, nuclear receptor coactivator; FTH1, ferritin heavy chain; GPX, glutathione peroxidase

Article Snippet: The primary antibodies used were rabbit antibodies specific for NCOA4 (1:1000; ab314553; Abcam, Cambridge, MA, USA), FTH1 (1:1000; CSB- PA008485 ; Cusabio Engineering Co., LTD, Wuhan, China), GPX4 (1:1000; ab125066; Abcam), and GAPDH (1:1000; ab8245; Abcam).

Techniques: Western Blot

Journal: Journal of Cardiothoracic Surgery

Article Title: NSUN2 inhibits NCOA4 expression to alleviate ferroptosis and inflammation in sepsis-induced myocardial injury in a m 5 C manner

doi: 10.1186/s13019-025-03554-z

Figure Lengend Snippet: NSUN2 inhibited NCOA4 expression in a m 5 C-dependent manner. A , The expression of NCOA4, FTH1, and GPX4 after NSUN2 overexpression in H9c2 cells was detected by RT-qPCR; B , MeRIP-qPCR assay was performed to detect m 5 C levels of NCOA4, FTH1, and GPX4 in vector and NSUN2 groups; C , RIP assay evaluated the interaction of NSUN2 and NCOA4 in H9c2 cells; D , Random Forest method was used to predict possible m 5 C sites of NCOA4; Dual-luciferase gene reporter assay evaluated the binding of NSUN2 and NCOA4 at sites E , 501, F , 1138, and G , 2917; H , RNA stability assay was used to detect the NCOA4 expression when actinomycin D treated at different time points (0, 4, 8, and 12 h) after NSUN2 overexpression NCOA, nuclear receptor coactivator; FTH1, ferritin heavy chain; GPX, glutathione peroxidase; NSUN, NOL1/NOP2/SUN domain; RT-qPCR, reverse transcription-polymerase chain reaction; MeRIP, Methylated RNA immunoprecipitation; RIP, RNA immunoprecipitation; m 5 C, 5-methylcytosine

Article Snippet: The primary antibodies used were rabbit antibodies specific for NCOA4 (1:1000; ab314553; Abcam, Cambridge, MA, USA), FTH1 (1:1000; CSB- PA008485 ; Cusabio Engineering Co., LTD, Wuhan, China), GPX4 (1:1000; ab125066; Abcam), and GAPDH (1:1000; ab8245; Abcam).

Techniques: Expressing, Over Expression, Quantitative RT-PCR, Plasmid Preparation, Luciferase, Reporter Assay, Binding Assay, Stability Assay, Reverse Transcription, Polymerase Chain Reaction, Methylation, RNA Immunoprecipitation

Journal: Journal of Cardiothoracic Surgery

Article Title: NSUN2 inhibits NCOA4 expression to alleviate ferroptosis and inflammation in sepsis-induced myocardial injury in a m 5 C manner

doi: 10.1186/s13019-025-03554-z

Figure Lengend Snippet: Overexpressing NCOA4 downregulated cell viability and upregulated LDH activity, inflammation, and ferroptosis in LPS-induced SIMI. A , NCOA4 mRNA level was detected by RT-qPCR; B , The cell viability of H9c2 cells in each group was analyzed by CCK-8 assay; C , Determination of LDH enzyme activity in each group; D , The contents of TNF-α, IL-6, and IL-8 in each group was evaluated by ELISA; Cellular E , Fe 2 + level, F , GSH content, and G , relative ROS production in each group were determined by commercial kits; H , The protein levels of NCOA4, FTH1, and GPX4 in each group was analyzed by Western blot RT-qPCR, reverse transcription-polymerase chain reaction; CCK-8, cell counting kit-8; LDH, lactate dehydrogenase; ELISA, enzyme-linked immunosorbent assay; TNF-α, tumor necrosis factor-α; IL, interleukin; Fe 2+ , ferrous iron; GSH, glutathione; ROS, reactive oxygen species; NCOA, nuclear receptor coactivator; FTH1, ferritin heavy chain; GPX, glutathione peroxidase

Article Snippet: The primary antibodies used were rabbit antibodies specific for NCOA4 (1:1000; ab314553; Abcam, Cambridge, MA, USA), FTH1 (1:1000; CSB- PA008485 ; Cusabio Engineering Co., LTD, Wuhan, China), GPX4 (1:1000; ab125066; Abcam), and GAPDH (1:1000; ab8245; Abcam).

Techniques: Activity Assay, Quantitative RT-PCR, CCK-8 Assay, Enzyme-linked Immunosorbent Assay, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Cell Counting