Effects of GCS on P-gp in NCI/ADR-RES Transfectants. (A) GCS and P-gp proteins detected by Western blot. Detergent-soluble protein (50 μg/lane) from NCI/ADR-RES (ADR-RES), NCI-ADR-RES/GCS (GCS) and NCI/ADR-RES/asGCS (asGCS) cells was immunoblotted with anti-GCS or anti-P-gp antibody. GAPDH was used as loading control. (B) Ceramide glycosylation catalyzed by GCS. Cells were incubated with NBD C6-Cer (100 nM) in 1% BSA RPMI-1640 medium, at 37°C for 2 hr. C6-Cer and C6-GlcCer were identified on chromatograms with commercial standard (St.) and measured using spectrophotometry. *, p < 0.001 compared to ADR-RES cells. (C) Immunostaining of GCS and P-gp. Cells were incubated with anti-human GCS (green) and anti-P-gp (red) following addition of Alexa 488- and Alexa 667-conjugated secondary antibodies. DAPI in mounting solution was used for nucleus counterstaining (blue). Ctrl, NCI/ADR-RES cells were incubated with the secondary antibodies alone, as specificity control; Fluo, merged fluorescence microphotograph (× 200). (D) Doxorubicin accumulation. After 1 hr incubation with doxorubicin (0.1 mg/ml), cellular doxorubicin was documented by fluorescence microscopy (× 200) and analyzed by HPLC, following methanol extraction. Doxorubicin amount was normalized to 100,000 cells. *, p < 0.001.