Fig. 5.

Itraconazole blocks C1GALT1-mediated malignant phenotypes and EGFR activity. a Effects of itraconazole on viability of SAS and OEC-M1 cells. SAS cells were transfected with empty pcDNA3.1 (Mock) or C1GLAT1/pcDNA3.1A (C1GALT1) and OEC-M1 cells were transfected with non-targeting siRNA (siCtr) or siRNA against C1GALT1 (siC1GALT1-1, siC1GALT1-2) for 48 h before seeding. Cells were treated with DMSO or 1.25 μM itraconazole (ITZ) after seeding for 24 h (Day 0). Viability was analyzed at day 4 using MTT assay. Data are analyzed by Student’s t-test. **P < 0.01. b Effects of itraconazole on cell migration and invasion. SAS cells were transfected with empty pcDNA3.1 (Mock) or C1GLAT1/pcDNA3.1A (C1GALT1) and OEC-M1 cells were transfected with non-targeting siRNA (siCtr) or siRNA against C1GALT1 (siC1GALT1-1, siC1GALT1-2) for 48 h before seeding. Cells were treated with DMSO or 1.25 μM itraconazole (ITZ) in the upper chamber of migration and invasion assays. Data are analyzed by Student’s t-test. *P < 0.05. **P < 0.01. (c) Effects of itraconazole on EGF-induced phosphorylation of EGFR and AKT. SAS cells were transfected with empty pcDNA3.1 (Mock) or C1GLAT1/pcDNA3.1A (C1GALT1) and OEC-M1 cells were transfected with non-targeting siRNA (siCtr) or siRNA against C1GALT1 (siC1GALT1-1, siC1GALT1-2) for 48 h before seeding. Cells were treated with DMSO, 1.25 μM or 2.5 μM itraconazole (ITZ) for 48 h. After starvation for 4 h, the cells were treated with (+) or without (−) EGF (10 ng/mL) for 5 min and collected for western blot analysis. GAPDH was an internal control. d Effects of itraconazole on Tn antigen expression on EGFR. SAS and OEC-M1 cells treated DMSO or 2.5 μM itraconazole (ITZ) for 48 h. Five hundred micrograms of proteins from cell lysates were used for each VVA pull-down analysis. EGFR, C1GALT1, and GAPDH from whole cell lysates were shown in the lower panels