Mixed-backbone oligonucleotide targeting human glucosylceramide synthase.
a. Cellular uptake of MBO. After exposure of cancer cells to Cy3-labeled MBO-asGCS (50 nM) for the indicated periods, cellular Cy3-MBO-asGCS was measured at λexcitation 550 nm and λemission 570 nm. b. Influence of MBOs on GCS expression. MBO-asGCS or MBO-SC was introduced into NCI/ADR-RE cells (ADR-RE) with Lipofectamine 2000 in Opti-EME I reduced-serum medium. After 48 hr growth, total RNA and protein were extracted. Total RNA (100 ng/reaction) was analyzed by RT-PCR. For Western blots, total protein (50 µg/lane) was subjected to 4–20% SDS-PAGE electrophoresis. Proteins were transferred to nitrocellulose and immunoblotted with GCS primary antibody (1∶500) and detected using ECL plus. GCS protein levels were presented as the ratios of the optical densities in GCS bands normalized against GAPDH.
