Lysis solution

Protein samples were extracted from myocardial tissue using a lysis solution (Beyotime Biotechnology, China). The samples were resolved by SDS–polyacrylamide gel electrophoresis, and transferred to polyvinylidene fluoride membranes (Merck Millipore, Billerica, MA, USA). All the blots were cut prior to hybridization with antibodies. Primary antibodies against SERCA2a (1:40,000) (Abcam, Cambridge, UK), α-fodrin (1:1000) (Abcam, Cambridge, UK), JPH2 (1:1000) ( Santa Cruz Biotechnology, Santa Cruz, CA, USA) and GAPDH (1:5000) ( Proteintech Group , China)were immuno-blotted overnight at 4℃. After this, membranes were immuno-blotted at room temperature for 1 h with corresponding secondary antibodies (1:5000) (ZSGB-BIO, Beijing, China). A chemiluminescence detection system (Tanon Imaging System, Tanon, Shanghai, China) was used to visualize protein bands. The data were analyzed on ImageJ 1.51 (NIH, USA).

Proteins were abstracted from liver tissue using a lysis solution (Beyotime, Shanghai, China) with 0.5 mM PMSF and phosphatase inhibitors. Protein concentrations were determined to calculate the loading amount. SDS-PAGE was performed on proteins using a 10% acrylamide gel. The specific operation process of protein immunoblotting also referred to a previous study [23 (link)]. Antibodies against t-Akt, p-Akt, SREBP-1c, and β-actin were from Beyotime Biotech, Shanghai, China. The secondary antibodies were from Proteintech Group, Inc., Wuhan, China.

Malondialdehyde (MDA), a product of membrane lipid peroxidation, shows a positive correlation with ferroptosis. A Lipid Oxidation (MDA) Assay Kit (S0131S; Beyotime) was used to detect MDA levels in SFs. Briefly, the cells were lysed using Lysis Solution (P0013; Beyotime) and centrifuged. The supernatant was collected and the protein concentration was detected using a BCA Protein Assay Kit (P0010; Beyotime). After preparing the MDA working solution in accordance with the kit instructions, a sample aliquot or blank control was added for determination. The test solution was boiled for 15 min, cooled to room temperature, and centrifuged to obtain the supernatant. Finally, the absorbance was measured at 532 nm using a multimode reader (Spark Cyto, TECAN).
C11-BODIPY 581/591 is a lipid-soluble fluorescent probe used to indicate lipid peroxidation in living cells. According to the manufacturer’s instructions, an appropriate volume of C11-BODIPY 581/591 (RM02821, ABclonal) was added to the cells and incubated for 30 min. After removal of the excess dye by washing with PBS, a cell pellet was obtained by 0.25% trypsin digestion and centrifugation. The cells were then resuspended in PBS containing 5% fetal bovine serum (FBS) and detected by flow cytometry (CytoFLEX LX, Beckman Coulter).

The cells were collected, and the total intracellular protein was extracted. Next, 80 μL of lysis solution (Beyotime) was added, and the sample was vortexed for sufficient lysis. The sample was centrifuged for 5 min (4 °C, 12,000 rpm), the supernatant was carefully aspirated and collected in another EP tube, and the total protein concentration was determined using the BCA method. After sample preparation, equal amounts of a sample protein (10 μg) were loaded onto an SDS–PAGE gel (10% separating gel, 5% stacking gel). After electrophoresis, the samples were transferred onto a nitrocellulose membrane. The membranes were blocked in blocking buffer for 1 h. After that, the membranes were incubated with primary antibodies against VEGF-A (Boster, Wuhan, China) and α-tubulin (Beyotime), followed by secondary antibodies coupled with horseradish peroxidase for 1 h. Reactive bands were detected with an ECL Western Blot Detection Kit (Boster) and visualized with a Tanon Imager program (Tanon, Shanghai, China). Non-saturated bands were selected to perform densitometric quantification using ImageJ software.

Renal tissue was treated with lysis solution (Beyotime Biotechnology, Shanghai, China) and ultrasonicated. Total protein was separated by SDS-PAGE and transferred onto 0.45-μm polyvinylidene difluoride membranes (Millipore, Billerica, MA, USA). After blocking in Tris-buffered saline containing 5% non-fat milk for 1 h at 25 °C, the membranes were incubated for 16–18 h at 4 °C with the following antibodies: anti-4-hydroxynonenal (4-HNE; 1:1000; Abcam), anti-caspase3 (1:1000; Cell Signalling Technology, Danvers, MA, USA), anti-Bcl-2 (1:1000; Abcam), anti-Bax, anti-catalase (CAT, 1:500; Sangon Biotech, Shanghai, China), anti-Nrf2 (1:1000; Abcam), anti-NQO1 (1:8000; Abcam), anti-heme oxygenase 1 (HO-1; 1:1000; Abcam), anti-SOD2 (1:500; Sangon Biotech), anti-Akt, anti-phospho-Akt, anti-PI3K, anti-phospho-PI3K, and β-actin (1:2000, Cell Signalling Technology). The membranes were subsequently incubated with the secondary antibody for 1 h at 25 °C, and protein bands were detected by electrochemiluminescence (Millipore). ImageJ software (NIH, Bethesda, MD, USA) was used to analyse the protein band intensities.