Gb3 Synthesis and β‐Catenin Recruitment Are Involved in MDR1 Transactivation. To silence Gb3 synthase, cells were transfected with siRNA-Gb3S (100 nM) or control siRNA (siRNA-SC) twice and grown in 10% FBS RPMI-1640 medium for 7 days. (A) MDR1 promoter activity. *, P < 0.001 compared with siRNA-SC. (B) Western blot. Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc. (C) Cellular efflux. *, p < 0.001 compared with siRNA-SC. (D) Immunostaining. Cells were incubated with anti-human Gb3 synthase (red) and anti-P-gp (green) following addition of Alexa 667- and Alexa 488-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Fluo., merged fluorescence microphotograph (x 200). (E) β-catenin/Tcf4 on P-gp expression. NCI/ADR-RES cells were exposed to FH535, β-catenin/Tcf4 inhibitor in 5% FBS medium for 24 hr.