Down-regulation of glucosylceramide synthase (GCS) affects Notch activation.

<p>(A) After lentivirus-mediated transduction with shRNA targeting GCS (GCS) or control shRNA (NT), MEFs stably expressing VSV-Dll1 were selected with puromycin before being assayed for actin and GCS mRNA by semi-quantitative RT-PCR. (B) Cells stably transduced with the GCS or the control shRNA were surface labeled on ice with CTXB-Cy3 prior to fixation. Images were acquired using a 20x objective. Scale bar 20 µm. (C) Whole cell extracts of cells transduced with the GCS or the control shRNA were analyzed by Western blot using Dll1 antibody. (D) The impact of GCS silencing on Notch activation was tested in a coculture assay. Cells described in panel A were cocultured with a U2OS line expressing the Notch1 receptor. Notch activation was evaluated using a CSL reporter strategy as described in Figure 7. Error bars represent standard variation. The presented result is representative of four independent experiments.