Ecl chemiluminescence kit

ASTA and disulfiram (TETD) were obtained from MedChemExpress (New Jersey, USA). The purity of these compounds was >95%. ECL chemiluminescence kit, DAPI fluoromount-GTM mounting medium, cell counting kit-8 (CCK-8), and dimethyl sulfoxide (DMSO) were obtained from YEASEN (Shanghai, China). Hoechst 33342 was purchased from Solarbio (Beijing, China). Fetal bovine serum (FBS) was obtained from ExCell Bio (Shanghai, China). RPMI medium modified (1640) was obtained from Hyclone, Thermo Scientific (MA, USA). A cell cycle staining kit was provided by Multi Sciences (Hangzhou, China). Antibodies against caspase 4, caspase 11, gasdermin D, GAPDH, and HRP-labeled goat antirabbit IgG (H + L) were obtained from Immunoway (Plano, TX, USA). Antibodies against cyclin A2 (CCNA2), cyclin B1 (CCNB1), and cyclin E1 (CCNE1) were purchased from HUABIO (Hangzhou, China).

2 × 10⁵ target cells were diluted with 2 mL of medium, inoculated in 6-well plates, and cultured for 2 days. The culture supernatant was collected after centrifugation at 12,000 g for 10 min and then concentrated 4-fold using a 3KD ultrafiltration centrifuge tube (Millipore). Cultured cells were lysed with 200 μL of RIPA lysis buffer (Solarbio) containing 1 mM PMSF on ice, and the lysed supernatant was collected by centrifugation at 12,000 g for 10 min at 4°C and the total protein concentration was determined by BCA protein assay kit (Beyotime).
The culture medium supernatant samples and cell lysis samples were boiled for 10 min after adding loading buffer, separated by 12% SDS-PAGE gel, and transferred to 0.22 μm PVDF membrane (Millipore) after electrophoresis. After blocking with 5% skimmed milk powder for 1 h, the membrane was incubated with primary antibody at 4°C overnight and secondary antibodies at room temperature for 1 hour, and then visualized and photographed using an ECL chemiluminescence kit (Yeasen). In this study, the following antibodies were used: anti-beta actin antibody from mouse (Abcam), HRP-conjugated Goat Anti-Mouse IgG (H + L) (Proteintech), HRP-conjugated Goat Anti-Human IgG (H + L) (Proteintech).