Ncoa4

The Western blot was conducted as previously described (Zhang et al., 2019 (link)). Total protein was extracted from myocardial tissues or cells by RIPA buffer containing protease and phosphatase inhibitor cocktail. Protein concentration was determined using Bradford assay. Equal amounts of proteins from each sample were subjected to 10% SDS-PAGE and transferred to PVDF membranes. After blocking with 5% BSA, blots were incubated with the following antibodies at 4°C overnight: ACSL4 (Abclonal, A14439), GPX4 (Abclonal, A1933), FTH1(CST, 4393), TfR1 (Abclonal, A5865), NCOA4 (Abclonal, A5695), and LC3 A/B (CST, 12,741). After three washes with TBST, these membranes were incubated with HRP-conjugated goat anti-rabbit IgG (Proteintech; SA00001–2). Immune complexes were visualized with enhanced chemiluminescence. β-tubulin (ProteinTech; 10068-1-AP) was used as the endogenous control. The optical density of bands was analyzed by ImageJ software.

H9c2 rat cardiomyocyte cell line was get from Wuhan Punosi Life Science and Technology Co., Ltd (Wuhan, China). DMEM low-sugar medium and FBS was purchased from HyClone (Logan, UT). The CCK-8 was purchased from Dojindo (Kumamoto, Japan). LDH, CK-MB, ROS, and GSH test kits were purchased from Nanjing Jiancheng Bioengineering Institute (Nanjing, China). Iron assay kit and GPX4 primary antibodies were purchased from Abcam (Cambridge, USA). Rabbit anti-rat primary antibodies DNMT-1 (catalog number 24206–1-AP), GPX4 (catalog number 67763-1-Ig), P62 (catalog number 18420-1-AP), Beclin-1 (catalog number 11306-1-AP), GAPDH (catalog number 60004-1-Ig), and Cy3- (catalog number SA00009-2) and FITC- (catalog number SA00003-2)-labeled goat anti-rabbit secondary antibodies were obtained from ProteinTech (Wuhan, China). Rabbit anti-rat primary antibodies NCOA4 (catalog number A5695) and FTH (catalog number A19544) were obtained from ABclonal (Wuhan, China). Second antibodies were purchased from LI-COR Biosciences (IRDye 800CW; catalog number 926-32219, LI-COR Corporate, USA). DNMT-1 inhibitor 5-aza-CdR were purchased from Selleck (Houston, USA). Lipofectamine 2000 was obtained from Invitrogen (San Diego, USA).

Total protein was extracted from RAW264.7 cells or alveolar macrophages according to published methods [60 (link)]. The membranes were subjected to an overnight incubation process at a controlled temperature of 4 °C, during which they were treated with primary antibodies. These antibodies were carefully diluted to a ratio of 1:1000 and included NCOA4 (Abclonal), GPX4 (Abclonal), FTH1 (ABclonal), LC3 (ABclonal) or β-Actin (ABclonal). The membranes were then incubated with secondary antibodies (diluted 1:1000, ABclonal, China) at room temperature for 1 h. Signals were detected using an advanced enhanced chemiluminescence system manufactured by Thermo Scientific, and relative protein expression was finally quantified with ImageJ software.