Journal: Frontiers in pharmacology

Article Title: Transcription factor MEF2D regulates aberrant expression of ACSL3 and enhances sorafenib resistance by inhibiting ferroptosis in HCC.

doi: 10.3389/fphar.2024.1464852

Figure Lengend Snippet: FIGURE 2 The aberrant expression of ACSL3 in HCC protects cells from ferroptosis. (A) Genes highly correlated with ACSL3 in HCC were analyzed using Pearson text (B, C) Heatmaps displayed the top 50 genes that are positively and negatively correlated with ACSL3 in HCC. (D) KEGG pathway analysis revealed the correlation between ACSL3 and fatty acid biosynthesis and ferroptosis signaling pathway (E, F) After transfecting ACSL3-oe and control plasmids into PLC/PRF/5 cells, the mRNA and protein expression levels of ACSL4, GPX4, and FTH1 were quantified using qPCR and Western blotting. Vinculin protein served as the internal control. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: ACSL3 antibody (Santa cruz, 1:100, Cat# sc-166374), ACSL4 Rabbit pAb (ABclonal, 1:1,000, Cat#A6826), MEF2D antibody (Santa cruz, 1:500, Cat# sc-271153), GPX4Monoclonal antibody (proteintech, 1:1,000, Cat No.: 67763-1- Ig), Ferritin heavy chain Polyclonal antibody (FTH1) (proteintech, 1:1,000, Cat No.: 11682-1-AP), Vinculin Monoclonal antibody (proteintech, 1:10,000, Cat No.: 66305-1-Ig).

Techniques: Expressing, Control, Western Blot

Journal: Oncology Letters

Article Title: Role of CYP4F2 as a novel biomarker regulating malignant phenotypes of liver cancer cells via the Nrf2 signaling axis

doi: 10.3892/ol.2020.11874

Figure Lengend Snippet: CYP4F2 overexpression inhibits the Nrf2 signaling pathway in liver cancer cells. (A) Reverse transcription-quantitative PCR was used to determine the Nrf2 mRNA expression levels in NC, Hep3B+vector and Hep3B+CYP4F2. (B) Western blot analysis was used to determine the protein expression levels in 5 genes and the results were quantified for CYP4F2 (C), Nrf2 (D), NQO1 (E), HO-1 (F) and FTH1 (G). Data are presented as the mean ± SD. One-way ANOVA was used with the Bonferroni correction. *P<0.05; **P<0.01; ***P<0.001. There is no significant difference between NC and Hep3B+vector in all figures. Hep3B cells were transfected with lentivirus to overexpress CYP4F2 (Hep3B+CYP4F2), or transfected with control lentivirus was used as a negative control (Hep3B+vector), and untreated Hep3B cells served as normal control (NC).

Article Snippet: Subsequently, the membrane was blocked using 5% skimmed milk and incubated overnight at 4°C with primary antibodies against CYP4F2 (cat. no. AF9051; 1:1,000; Affinity Biosciences), Nrf2 (cat. no. 66504-1-Ig; 1:1,000; ProteinTech Group, Inc.), NQO1(cat. no. 67240-1-Ig; 1:5,000; ProteinTech Group, Inc.), HO-1 (cat. no. 66743-1-Ig; 1:1,000; ProteinTech Group, Inc.) and FTH1 (cat. no. 10727-1-AP; 1:1000), GAPDH (cat. no. BA2913; 1:500; Wuhan Boster Biological Technology, Ltd.) as the internal reference.

Techniques: Over Expression, Reverse Transcription, Real-time Polymerase Chain Reaction, Expressing, Plasmid Preparation, Western Blot, Transfection, Control, Negative Control

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

Journal: Molecular Medicine Reports

Article Title: Salusin-β participates in high glucose-induced HK-2 cell ferroptosis in a Nrf-2 -dependent manner

doi: 10.3892/mmr.2021.12313

Figure Lengend Snippet: Effects of salusin-β knockdown on HG-induced ferroptosis in HK-2 cells. HK-2 cells in logarithmic growth were transduced with lentivirus particles carrying salusin-β shRNA or negative control shRNA at MOI=50 for 48 h and then challenged by HG stimulation for another 48 h. (A) Representative blot images and (B) quantitative analysis of GPX4, SLC7A11, FTH-1 and TFR-1 . (C) Relative mRNA levels of GPX4, SLC7A11, FTH-1 and TFR-1 . (D) Iron contents. (E) GSH contents. (F) MDA levels. (G and H) ROS generation was assessed by dichloro-dihydro-fluorescein diacetate fluorescence. (I) The proliferation of HK-2 cells was evaluated by EdU staining. (J) The cell proliferation was assessed by CCK-8 assay. (K) The cytotoxicity was determined by LDH release assay. Scale bar=200 µm. Data are presented as the mean ± SD (n=4–5 per group). *P<0.05 vs. Con shRNA. † P<0.05 vs. HG + Con shRNA. HG, high glucose; NG, normal glucose; sh, short hairpin RNA; MOI, multiplicity of infection; GPX4 , glutathione peroxidase 4; SLC7A11 , solute carrier family 7 (cationic amino acid transporter, y+ system) member 11; FTH-1 , ferritin heavy polypeptide 1; TFR-1 , transferrin receptor 1; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; LDH, lactate dehydrogenase; Con, control.

Article Snippet: SA00001-1 and SA00001-2) were obtained from ProteinTech Group, Inc. Antibodies against glutathione peroxidase 4 ( GPX4 ; cat. no. 52455), solute carrier family 7 (cationic amino acid transporter, y+ system) member 11 ( SLC7A11 ; cat. no. 12691), ferritin heavy polypeptide 1 ( FTH-1 ; cat. no. 4393), transferrin receptor 1 ( TFR-1 ; cat. no. 13113) and Nrf-2 (cat. no. 12721) were purchased from Cell Signaling Technology, Inc.

Techniques: Transduction, shRNA, Negative Control, Fluorescence, Staining, CCK-8 Assay, Lactate Dehydrogenase Assay, Infection

Journal: Molecular Medicine Reports

Article Title: Salusin-β participates in high glucose-induced HK-2 cell ferroptosis in a Nrf-2 -dependent manner

doi: 10.3892/mmr.2021.12313

Figure Lengend Snippet: Effects of salusin-β overexpression on HG-induced ferroptosis in HK-2 cells. HK-2 cells were transduced with lentivirus expressing salusin-β vectors or empty vectors (MOI=50) for 48 h before administration of NG or HG for another 48 h. (A) Representative blot images and (B) quantitative analysis of GPX4, SLC7A11, FTH-1 and TFR-1 . (C) Relative mRNA levels of GPX4, SLC7A11, FTH-1 and TFR-1 . (D) Iron contents. (E) GSH contents. (F) MDA levels. (G and H) ROS generation was assessed by dichloro-dihydro-fluorescein diacetate fluorescence. (I) The proliferation of HK-2 cells was evaluated by EdU staining. (J) The cell proliferation was assessed by CCK-8 assay. (K) The cytotoxicity was determined by LDH release assay. Scale bar=200 µm. Data are presented as the mean ± SD (n=4–5 per group). *P<0.05 vs Vector. † P<0.05 vs. HG + Vector. HG, high glucose; MOI, multiplicity of infection; NG, normal glucose; sh, short hairpin RNA; GPX4 , glutathione peroxidase 4; SLC7A11 , solute carrier family 7 (cationic amino acid transporter, y+ system) member 11; FTH-1 , ferritin heavy polypeptide 1; TFR-1 , transferrin receptor 1; GSH, glutathione; MDA, malondialdehyde; ROS, reactive oxygen species; LDH, lactate dehydrogenase; Con, control; OE, overexpression.

Article Snippet: SA00001-1 and SA00001-2) were obtained from ProteinTech Group, Inc. Antibodies against glutathione peroxidase 4 ( GPX4 ; cat. no. 52455), solute carrier family 7 (cationic amino acid transporter, y+ system) member 11 ( SLC7A11 ; cat. no. 12691), ferritin heavy polypeptide 1 ( FTH-1 ; cat. no. 4393), transferrin receptor 1 ( TFR-1 ; cat. no. 13113) and Nrf-2 (cat. no. 12721) were purchased from Cell Signaling Technology, Inc.

Techniques: Over Expression, Transduction, Expressing, Fluorescence, Staining, CCK-8 Assay, Lactate Dehydrogenase Assay, Plasmid Preparation, Infection, shRNA

Journal: Molecular Medicine Reports

Article Title: Salusin-β participates in high glucose-induced HK-2 cell ferroptosis in a Nrf-2 -dependent manner

doi: 10.3892/mmr.2021.12313

Figure Lengend Snippet: Expression of salusin-β during the process of ferroptosis. (A) HK-2 cells were treated with ferroptosis activators for 24 h, including erastin (5 µM), GPX4 inhibitors (1 µM for RSL3 and 5 µM for FIN56) and GSH synthase inhibitor (100 µM for buthionine sulfoximine), the protein expression of salusin-β was analyzed by western blotting. (B) HK-2 cells were pretreated with ferrostatin-1 (1 µM) for 30 min and incubated with HG for 48 h, the protein expression of salusin-β was then detected by western blotting. (C) The action mechanism of salusin-β involved in ferroptosis in diabetic kidneys via inactivation of Nrf-2 signaling. Data are presented as the mean ± SD (n=5 per group). *P<0.05 vs. Veh or NG. † P<0.05 vs. HG. GPX4, glutathione peroxidase 4; GSH, glutathione; Nrf-2 , nuclear factor erythroid-derived 2-like 2; NG, normal glucose; HG, high glucose; Veh, Vehicle SLC7A11, solute carrier family 7 (cationic amino acid transporter, y+ system) member 11; FTH-1, ferritin heavy polypeptide 1; TFR-1, transferrin receptor 1; ROS, reactive oxygen species.

Article Snippet: SA00001-1 and SA00001-2) were obtained from ProteinTech Group, Inc. Antibodies against glutathione peroxidase 4 ( GPX4 ; cat. no. 52455), solute carrier family 7 (cationic amino acid transporter, y+ system) member 11 ( SLC7A11 ; cat. no. 12691), ferritin heavy polypeptide 1 ( FTH-1 ; cat. no. 4393), transferrin receptor 1 ( TFR-1 ; cat. no. 13113) and Nrf-2 (cat. no. 12721) were purchased from Cell Signaling Technology, Inc.

Techniques: Expressing, Western Blot, Incubation, Derivative Assay