Fig. 2: MLN8237 treatment activated the calpain pathway and induced apoptosis through p27 degradation and Bax cleavage in AGS cell line.

a MLN8237 treatment led to p27 downregulation and Bax cleavage. AGS cells were cultured at the indicated concentration of MLN8237 for 72 h and incubated time at 200 nM. The protein samples were subjected to an immunoblotting analysis of p21, p27, c-Bax, c-PARP, and Actin. b Calpain knockdown attenuated the p27 turnover and Bax cleavage induced by the treatment of the cells with 200 nM MLN8237 for 72 h. Calpain 4 was knocked down and treated with 200 nM MLN8237 for 72 h in AGS. Protein expression was determined by immunoblotting analysis. c Immunoblotting analysis demonstrated that both calpain 1 and calpain 2 degraded p27 and cleaved Bax. Calpain 1/4 or calpain 2/4 were overexpressed in AGS gastric cancer cells and then treated with MLN8237 at 200 nM, and subjected to immunoblotting analysis for indicated protein expression. d Calpain 1 or calpain 2 enhanced apoptosis of MLN8237-treated AGS gastric cancer cells. Calpain 1/4 or calpain 2/4 were overexpressed and treated with 200 nM MLN8237 for 72 h. Apoptosis was determined by FCM. The experiment was performed independently three times. e Both p27 and Bax combined with calpain. Calpain 1/4-Flag was overexpressed with pCMV-p27 or Bax-HA plasmids for 24 h in 293 T. Equivalent cell lysate was immunoprecipitated with Flag beads and immunoblotted p27 and HA protein levels. f, g MLN8237-induced Ca²⁺ release to the cytoplasm. AGS was treated with MLN8237 for 72 h at indicated concentration and loaded with 5 μM Fluo-3/AM for 30 min at 37 °C. The labeled cells were analysis by FCM (f). g AGS was pre-incubated with 1 and 2 uM EDTA for 2 h and followed treated with 200 nM MLN8237 for 72 h. Error bars represent SD from three independent experiments. Asterisk (*) indicates a significant difference. **P < 0.01, ***P < 0.001, ****P < 0.001, two-tailed Student’s t test