Silencing GCS Represses MDR1 Expression by Decreasing cSrc/β-Catenin Signaling. After MBO-asGCS treatments (0, 50, 100, 200 nM), drug resistant NCI/ADR-RES cells were cultured in 10% FBS RPMI-1640 medium for 7 days. The NCI/ADR-RES cells were incubated with verapamil (10 μg, 2 hr) in 5% FBS RPMI-1640 medium to inhibit P-gp function. (A) Western blots. Equal amounts of total cellular proteins or nuclear proteins (50 μg/lane) were resolved by 4-20% gradient SDS-PAGE and immunoblotted with indicated primary antibodies. GD3 syn, GD3 synthase; 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 MBO-asGCS treatments. *, p < 0.001 compared with vehicle. (C) Paclitaxel accumulation and efflux. Cells were incubated with Flutax-2 (0.5 μM) in medium at 37°C for 2 hr to measure paclitaxel accumulation (top panel). After washing with ice-cold PBS, cells were incubated with fresh medium for an additional 2 hr to measure paclitaxel efflux (bottom panel). *, p < 0.001 compared with vehicle treatment.