Fig. 2

Knockout of CALM1 and treated with EGFR inhibitor markedly impairs the proliferation, apoptosis and cell cycle of ESCC cells. a Basal expression of CALM1 and EGFR on mRNA by qRT-PCR was detected in three tested ESCC cell lines. b qRT-PCR showing remarkable silencing efficiency in three ESCC cell lines infected with sgCALM1-1, sgCALM1-2 and sgCALM1-3. c Comparison of the proliferation of ESCC cell lines after knockout of CALM1 with or without inhibition of EGFR by MTT. Cells were pretreated with the inhibitors for 1 h and maintained in culture. d Influence of CALM1 and EGFR inhibitor on apoptosis in ESCC cells, analyzed by using flow cytometry. The percentage of Annexin V-FITC-positive cells to the total cells is shown in bar graphs. e Flow cytometry analysis of the effects of EGFR inhibitor treatment on cell cycle in ESCC after CALM1 knockout. All assays were performed in triplicate and the results are presented as the mean ± S.D. in all panels (c, d, e). CALM1, calmodulin1; EGFR, epidermal growth factor receptor. ESCC, esophageal squamous cell carcinoma; MTT, 3-(4, 5-dimethyl-2-thiazolyl)-2,5-diphenyl-2H-tetrazolium bromide; sgRNA, guide RNA. *P < 0.05, **p < 0.01, ***p < 0.001(Student's t-test)