Anti cleaved caspase 7 antibody

The 3-acyl isoquinolin-1(2H)-one complex 4f was synthesized according to a known procedure developed in our previous work. Cell counting kits-8 (CCK8) was purchased from Beyotime Biotechnology, dimethyl sulfoxide (DMSO) and RIPA lysis buffer were purchased from Sigma (Beverly, MA, USA). Primary antibodies such as anti-caspase3 antibody (cat#14220T), anti-cleaved-caspase9 antibody (cat#52873), anti-cleaved-caspase7 antibody (cat#8438), anti-parp antibody (cat#9542), anti-bcl-2 antibody (cat#15071T), anti-bax antibody (cat#5023), anti-β-tubulin antibody (cat#2128s), anti-ERK1/2 antibody (cat#4695T), anti-p-ERK1/2 antibody (cat#4370T), anti-MEK1/2 antibody (cat#9126s), anti-p-MEK1/2 antibody (cat#9154T), (HRP)-labeled anti-rabbit secondary antibody (Cat#7074) and (HRP)-labeled anti-mouse secondary antibody (Cat#7076) were purchased from Cell Signaling Technology. Anti-GSDME antibody (ab215191), anti-GSDMD antibody (ab210070), anti-p-MLKL antibody (ab187091) and anti-MLKL antibody (ab184718) were purchased from Abcam. Anti-CDK1 antibody (AF1516) was purchased from Beyotime Biotechnology.

Cells were plated in 96-well clear bottom black plates at 1 × 10⁴ cells per well and allowed to adhere for 24 h. Cells were treated with the respective concentration of flavonoids and incubated for 48 h. Medium was removed and cells were fixed at room temperature for 15 min with 4% paraformaldehyde, rinsed three times with PBS, and permeabilised at room temperature for 5 min with 0.1% Triton X-100. Then, non-specific binding was blocked with 10% heat-inactivated goat serum in PBS for 1 h at room temperature. Cells were incubated with the following antibodies at 4°C, overnight: anti-p53 antibody (sc-126, 1:200) and anti-Nrf2 antibody (sc-365949, 1:200) obtained from Santa Cruz Biotechnology, and anti-cleaved-caspase-3 antibody (#9664, 1:200) and anti-cleaved-caspase-7 antibody (#8438, 1:200) purchased from Cell Signaling Technology. After three washes with PBS, cells were incubated with Alexa Fluor 594 conjugated antibody (A11032 or A11037; Invitrogen) for 1 h in the dark. Cell nuclei were stained with Hoechst 33342 (Invitrogen) and F-actin was stained with Phalloidin-Atto 488 (Sigma-Aldrich). Fluorescent signals were examined and captured using a BD Pathway 855 confocal microscope (Becton Dickinson). The mean fluorescence intensity was calculated using ImageJ software (National Institutes of Health).