Caspase8

Cells from each time group were added 100 μL of cell buffer and collected in 1.5 mL EP tubes. Protein concentration in the samples was quantified using the BCA method. The samples were heated in a 100 °C water bath for 5 min to denature the proteins. For each sample, 20 μg of protein was subjected to polyacrylamide gel electrophoresis. The electrophoresis was conducted at 90 V for 90 min, followed by transferring the target bands from the gel to a PVDF membrane under ice-water bath conditions. The primary antibody solutions: NSE (1:1000; EPITOMICS, USA), GRP94 (1:100; Affinity, USA), GHOP, Fas, FasL, TNFR1, TNF-α, DR5, TRAIL and Caspase8 (all 1:1000; Affinity, USA), Caspase3 (1:100; Beijing Zhongshan, China) and β-actin (1:5000; rabbit monoclonal antibody, China) were added and incubated overnight at 4 °C, and the membranes were washed with TBST. Corresponding goat anti-rabbit/mouse secondary antibody (1:5000; Beijing Zhongshan Golden Bridge, China) was added and incubated, followed by washing with TBST. The bands were scanned using a scanner, and the optical density of each band was analyzed using Image J software. Each experiment was repeated three times.

The uninduced, pre-induced, and induced cells for 1 h, 3 h, 5 h, and 8 h were crawled according to the standard IHC procedure. The cells were permeabilized with 0.1% Triton-X-100 for 10 min, followed by incubation with 3% H₂O₂ for 10 min. They were incubated with diluted primary antibodies: NSE (1:100; EPITOMICS, USA), GRP94 and GHOP (all 1:100; Affinity, USA), Fas, FasL TNFR1, TNF-α, DR5, TRAIL and Caspase8 (all 1:200; Affinity, USA), Caspase3 (1:100; Beijing Zhongshan Golden Bridge, China) overnight at 4 °C. Cells were then incubated with goat anti-rabbit/mouse polyclonal secondary antibody (1:200; Beijing Zhongshan Golden Bridge, China) at 37 °C for 30 min. The cells were then developed with 3,3′-diaminobenzidine (DAB) (Beijing Zhongshan Golden Bridge, China) and stained. Under light microscope (Olympus, Japan) high magnification ( × 100). Positive expression cells were identified by the presence of brown-yellow coloration in the cytoplasm or cell membrane, while cells without any coloration were considered negatively expressing cells. This count was performed 5 times in different fields for each sample, and 3 samples were observed in total.