Itraconazole inhibits HER2/PI3K signaling in esophageal cancer. A, Western blot analysis of p-PI3K, PI3K, PTEN, HER2, p-AMPK, and AMPK expression in itraconazole or DMSO-treated OE33, FLO-1, KYSE70 and KYSE510 cells. GAPDH is used as a loading control. B, RT–qPCR analysis of ERBB2 mRNA levels in OE33, FLO-1, KYSE70 and KYSE510 cells treated with 2.5 μM itraconazole or DMSO for 48 hours. Values represent the mean fold change ± SEM for three experiments relative to GAPDH. *, p<0.05 and **, p<0.01 versus control group by Student’s t-test. C, Western blot analysis of p-AKT and AKT expression in OE33 and KYSE510 cells treated with DMSO, 2.5 μM or 5 μM lapitinib for 48 hours. GAPDH is used as a loading control. D, Cell proliferation assay for OE33 and KYSE510 cells treated with 2.5 μM lapatinib. Cell numbers were counted at the indicated timepoints. The data are presented as the means ± SEM of three independent experiments. E, Western blot analysis of HER2, p-AKT, and AKT expression in OE33 and KYSE510 cells transfected with HER2-specific or NTC siRNAs for three days. GAPDH is used as a loading control. F, AlamarBlue-based cell proliferation assay for OE33 and KYSE510 cells treated with HER2-specific or NTC siRNAs for three days. Values represent the mean ± SEM for 8 wells. **, p<0.01 versus control group by Student’s t-test.