Fig. 4.

Identification of itraconazole as a C1GALT1 inhibitor. a Molecular docking analysis of itraconazole and C1GALT1. Right panel indicates the enlarged binding surface of C1GALT1 and itraconazole. b Effects of itraconazole on Tn antigens of the cell surface. Flow cytometry with FITC-VVA on surfaces of SAS, OEC-M1, and FaDu cells treated with solvent control DMSO or 2.5 μM itraconazole (ITZ) for 48 h. c Western blot analysis of Tn antigens on cellular proteins and C1GALT1 in SAS, OEC-M1, and FaDu cells treated with DMSO or 2.5 μM itraconazole (ITZ) for 48 h. GAPDH was an internal control. d Effects of itraconazole on C1GALT1 protein levels at various concentrations, as indicated. SAS and OEC-M1 cells were treated with DMSO or itraconazole (ITZ) for 48 h and collected for western blot analysis. e Degradation pathways of C1GALT1 mediated by itraconazole. Cells were treated with 2.5 μM itraconazole (ITZ) for 24 h, and then incubated with 10 μM chloroquine or 20 μM MG132 for 6 h, as indicated. C1GALT1 levels were analyzed by western blot analysis. f Effects of itraconazole on ubiquitination of C1GALT1. SAS and OEC-M1 cells were transfected with C1GALT1-HA/DNA3.1A for 48 h and then treated with 2.5 μM itraconazole (ITZ) and 20 μM MG132 for 6 h. Cell lysates were immunoprecipitated (IP) with anti-HA antibody and then immunoblotted (IB) with anti-ubiquitin antibody. C1GALT1-HA in whole cell lysates (input) was shown. g Effects of itraconazole on thermal stability of C1GALT1 analyzed using cellular thermal shift assays. Left panel, SAS and OEC-M1 cells were treated with DMSO or 2.5 μM itraconazole (ITZ) for 2.5 h. Cell lysates were incubated at room temperature (RT), 53, 55, or 57 °C for 3 min, followed by cooling at RT for 3 min. Right panel, cells were treated with DMSO or itraconazole (ITZ) at different concentrations, as indicated, for 2.5 h and cell lysates were incubated at 55 or 57 °C. GAPDH was the internal control. (−) unstained cells