R1210 1

Treated cells were harvested in radioimmunoprecipitation assay buffer (RIPA; Beyotime, P0013B) supplemented with protease inhibitor (Roche, 04693159001) and phenylmethylsulfonyl fluoride (PMSF; Beyotime, ST506) on ice, and the protein concentration was determined by using a BCA Protein Assay Kit (Thermo Scientific, 23250). Cellular proteins were separated via SDS-PAGE through 8–12% gels before they were transferred to polyvinylidene fluoride (PVDF) microporous membranes (EMD Millipore, IPVH00010). The blots were probed for 12 h at 4 °C with primary antibodies, and secondary antibodies were incubated with the PVDF membrane for 1 h at room temperature. Visualization was performed with a Molecular Imaging System (Carestream Health, INC., NY, USA). The antibodies used are as follows: anti-SLC40A1 rabbit antibody (Abcam, ab78066), anti-GCLM rabbit antibody (HuaBio, ET1705-87), anti-GCLC rabbit antibody (HuaBio, ET1704-38), anti-IκBα rabbit antibody (HuaBio, ET1603-6), anti-phospho-IκBα rabbit antibody (HuaBio, ET1609-78), anti-NF-kB p65 rabbit antibody (HuaBio, ET1603-12), anti-SLC7A11 rabbit antibody (Abcam, ab175186), anti-c-Jun rabbit polyclonal antibody (Proteintech, 24909-1-AP), anti-ferritin rabbit monoclonal antibody (Abcam, ab75973), anti-GAPDH rabbit antibody (HuaBio, R1210-1), and anti-β-actin antibody (HuaBio, 1102-1).

Transfected ECA-109 and TE-1 cells were lysed using precooled RIPA lysis buffer (Beyotime, China), and the protein concentration was quantified with the BCA Protein Assay Kit (Beyotime, China). Sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE, 10%) was employed to separate the proteins, and then the proteins were transferred to polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA). The membranes were blocked with 5% skim milk in a TBST solution for 1 h at room temperature and incubated with primary antibodies against Caspase 3 (1:500; ab13847, Abcam), Caspase 9 (1:2,000; ab32539, Abcam), Bax (1:1,000; ab32503, Abcam), Bcl-2 (1:2,000; ab182858, Abcam), E-cadherin (1:500; ab15148, Abcam), Vimentin (1:1,000; #12826, Cell Signaling Technology, Boston, USA), N-cadherin (1:1,000; #14215, Cell Signaling Technology, Boston, USA), and GAPDH (1:10,000; R1210-1, HuaBio, Hangzhou, China) at 4 °C overnight. Then, the membranes were incubated with an appropriate HRP-conjugated secondary antibody at room temperature for 2 h. Finally, the bands were visualized by enhanced chemiluminescence (Solarbio, China), and the intensity of the protein bands was analyzed using ImageJ software.