Cell counting kits 8

Firstly, HCC-LM3 and Hep3B cells were seeded into 96-well plates. The cells were collected and used Cell Counting Kits-8 (Beyotime, China) according to the manufacturer’s protocol. The absorbance was measured at OD450. Secondly, HCC cells were collected and fixing with ice cold acetone. After washing and blocked with BSA buffer (1% BSA, 0.3% Triton, 1 × PBS (Solarbio, China), the cells were incubated with KI67 (1:1000, CST, USA) antibody at 4 °C overnight. After washing with PBS, cells were incubated with corresponding fluorescent secondary antibody, and counterstained for nuclei using 4′, 6-diamidino-2-phenylindole (DAPI) (Solarbio, China). All experiments were performed in triplicate.

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.