Af5343

Total protein samples from cardiac tissue or H9c2 cells were obtained with RIPA lysis buffer (strong) containing 1% protease inhibitors and protein concentrations were determined. After concentration determination, proteins were separated by SDS-PAGE (7.5–12.5%) and transferred to PVDF membranes. The membranes were blocked with 5% skim milk and incubated overnight at 4 °C with primary antibodies Nrf2 (A21176, ABclonal), HO-1 (AF5393, Affinity), TfR1 (AF5343, Affinity), GPX4 (DF6701, Affinity), NOX4 (DF6924, Affinity), ABCB8 (A2653, ABclonal), FXN (DF6590, Affinity) (antibody dilution ratio 1: 1000) and β-tubulin (1:3000, AF7011, Affinity), respectively. The membranes were then incubated with the secondary antibody HRP Goat Anti-Rabbit IgG (H+L) (1:5000, AS014, ABclonal) for 2 h at room temperature. Protein bands detected by an enhanced chemiluminescence system were analyzed using ImageJ software.

OS cells were lysed with RIPA buffer and centrifuged at 12000 rpm for 15 min to collect the supernatant. The protein samples were separated by 10% SDS-PAGE and transferred to PVDF membranes (Millipore, Billerica, MA, USA). The PVDF membranes were blocked with 5% skim milk for 1.5 h and then incubated overnight with primary antibody at 4°C. After being washed with TBST 3 times (15 min each), the membranes were incubated with an HRP-conjugated secondary antibody at room temperature for 1 h. The protein bands were visualized with an Amersham Imager 600. Primary antibodies against Beclin-1 (ab207612, Abcam), LC3 (ab192890, Abcam), P62 (ab109012, Abcam), γ-H2AX (ab81299, Abcam), GPX4 (DF6701, Affinity), TFR (AF5343, Affinity), and NCOA4 (DF4255, Affinity) were diluted 1 : 1000; an antibody against GAPDH (#5174, CST, USA) was diluted 1 : 2000.

OS cells were seeded in 24-well plates at a density of 5 × 10⁴ cells per well and treated with the indicated drugs for 24 h. Then, the cells were fixed in 4% paraformaldehyde for 20 min and incubated with a primary antibody against LC3 (ab192890, Abcam), γ-H2AX (ab81299, Abcam), and TFR (AF5343, Affinity) at 4°C overnight. The cells were washed with PBS three times for 3 min each and then incubated with secondary antibodies for 1 h at room temperature. DAPI was used to stain the nuclei for 1 min. The samples were sealed and observed under a fluorescence microscope.