Anti caspase 9

Tumors excised from mice were fixed in 10% formalin, embedded in paraffin, and cut into 4-mm sections. Deparaffinized tumor sections were treated with 3% H₂O₂ for 10 min to block endogenous peroxidases and incubated with 5% blocking serum (goat serum) at room temperature for 30 min. After blocking, the slides were incubated with polyclonal rabbit anti-Caspase-3 (1:200 dilution; Proteintech, America), anti-Caspase-9 (1:100 dilution; Proteintech, Wuhan, China), anti-Bcl-2 (1:200 dilution; Proteintech, America), anti-Bax (1:200 dilution; Proteintech, Wuhan, China), and anti-Cytc (1:250 dilution; ABCAM, Britain) overnight at 4°C. The sections were then incubated for 1 h with a biotin labeled secondary antibody, followed by avidin-peroxidase reagent and 3,3ʹ-diaminobenzidine (DAB; Fuzhou, China) substrate. After hematoxylin counterstaining and dehydration, the sections were sealed with cover slips. Finally, the stained setions were observed under a microscope.

Cells transfected with either control siRNA or siR-β-catenin were cultured with or without cisplatin for 24 h. Subsequently, the cells were lysed with radioimmunoprecipitation assay lysis buffer (cat. no. P0013B; Beyotime Institute of Biotechnology) with protease and phosphatase inhibitors. Protein content was determined by a Bradford protein kit (cat. no. P0012S; Beyotime Institute of Biotechnology). The proteins (30 µg/lane) were separated by 10% SDS-PAGE (cat. no. P0012A; Beyotime Institute of Biotechnology) and transferred onto PVDF membranes (EMD Millipore). Following blocking with 5% non-fat milk for 1 h at room temperature, the membranes were incubated overnight at 4°C with the following antibodies: Anti-β-catenin (cat. no. 17565-1-AP; 1:4,000; ProteinTech Group, Inc.), anti-c-Myc (cat. no. 10828-1-AP; 1:2,000; ProteinTech Group, Inc.), anti-cyclin D1 (cat. no. 26755-1-AP; 1:1,000; ProteinTech Group, Inc.), anti-caspase 3 (cat. no. 19677-1-AP; 1:600; ProteinTech Group, Inc.), anti-caspase 9 (cat. no. 10380-1-AP; 1:800; ProteinTech Group, Inc.) and anti-β-actin (cat. no. 20536-1-AP; 1:800; ProteinTech Group, Inc.), followed by horseradish peroxidase-conjugated AffiniPure donkey anti-rabbit IgG (H+L) (cat. no. SA00001-9; 1:4,000; ProteinTech Group, Inc.) at 4°C for 2 h. The band intensity was tested using ImageJ v.1.47 software (National Institutes of Health).

Total protein was extracted using a bicinchoninic acid (BCA) assay kit by lysing cell precipitates with RIPA lysis buffer for 10–20 min on ice or at 4°C, followed by centrifugation at 12 000 × g for 15 min at 4°C. The protein lysate was diluted in 5X loading buffer and denatured by boiling at 100°C for 5–10 min. Protein samples (60 μg) were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to a polyvinylidene difluoride (PVDF) membrane by membrane transfer on ice at 250 mA. They were blocked with 5% nonfat milk for 1–2 h followed by incubation with primary antibodies overnight at 4°C in an orbital shaker. The membrane was incubated with goat anti-rabbit or anti-mouse IgG secondary antibody for 1–2 h at 4°C on an orbital shaker after primary antibody was washed off. Finally, the membrane was visualized with a chemiluminescence imager and exposed to the enhanced chemiluminescence (ECL) dye solution.
Primary antibodies used included: anti-SQLE (Proteintech, Cat# 12544–1-AP), anti-β-actin (BIOX, Cat# BX-009), anti-caspase 3 (CST, Cat# 9662S), anti-cleaved caspase 3 (CST, Cat# 9664T), anti-caspase 9 (Proteintech, Cat# 10380–1-AP), anti-GPX4 (CST, Cat# 52455S), anti-xCT (abcam, ab175186), anti-Bcl2 (CST, Cat# 15071T), and anti-BAX (CST, Cat# 5023T).