GCS Upregulates MDR1 Expression through Enhanced cSrc/β-Catenin Signaling. After a series of transient GCS transfection (0, 2.0, 4.0, 8.0 μg of pcDNA 3.1-GCS plasmid DNA in 100-mm dish), OVCAR-8 cells were cultured in 10% FBS RPMI-1640 medium for 7 days. OVCAR-8 cells transfected with pcDNA 3.1-GCS (8 μg) were then treated with 10 μM PP2 for 24 hr (+PP2). (A) Western blots. Equal amounts of detergent-soluble total cellular proteins or nuclear proteins (50 μg/lane) were resolved on 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. Gb3 syn, Gb3 synthase; p-cSrc, phosphorylated cSrc; p-FAK, phosphorylated FAK; p-β-catenin, phosphorylated β-catenin. (B) MDR1 expression. MDR1 promoter activity (top panel) and P-gp protein (bottom panel) were assessed as described in Methods, after 7 days of GCS transient transfection in OVCAR-8 cells. *, p < 0.001 compared with mock transfection; **, p < 0.001 compared with vehicle treatment in cells transfected with GCS (8 μg DNA). (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax‐2 (0.5 μM) in medium at 37°C. Accumulation of paclitaxel (top panel) was measured after 2 hr incubation. After washing with ice-cold PBS, cells were re-incubated with fresh medium for an additional 2 hr to measure efflux (bottom panel). *, p < 0.001 compared with the mock transfection. **, p < 0.001 compared with vehicle treatment in cells transfected with GCS (8 μg DNA).