Dll1AG does not recycle and exhibits a shorter half-life than wild type Dll1.

<p>(A) Surface proteins of Dll1- and Dll1AG-expressing OP9 cells were labeled with biotin and a recycling assay was performed as described in Materials and Methods. NDB: not debiotinylated; Mesna2: second Mesna treatment. (B) Western blot analysis of cells expressing Dll1 or Dll1AG. Approximate equal loading of the full-length form was used in order to facilitate estimation of the extent of metalloprotease cleavage. (C) HeLa cells were transiently-transfected with plasmids encoding Dll1-Apa or Dll1-Apa-AG together with GFP. Following cycloheximide treatment the levels of ligands were monitored as described in Figure 2. Western blots were performed using anti-Dll1 and anti-GFP antibodies.</p

Internalization of Dll1AG.

<p>Hela cells were transiently-transfected with VSV-Dll1 or VSV-Dll1AG. Cy3-coupled anti-VSV antibody uptake (for 0 or 30 minutes), and secondary labeling with Alexa 488-coupled anti-IgG (to label Dll1 present at the cell surface) were performed as described in the legend to Figure 3. The presented image is typical of the results obtained in multiple experiments. The graph at the bottom of the figure presents the quantitation of the abovementioned experiment, in which the proportion of wt or mutant ligand remaining at the cell surface is plotted against time. For each condition, 20 images were quantified. Values on the graph are indicated +/- standard deviation. The p-value was calculated using MATLAB. Scale bar, 10 µm.</p

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.</p

Dll1AG does not activate Notch signaling.

<p>U2OS cells, stably expressing HA-tagged Notch1, were transiently-transfected with a CSL-luciferase construct and pRL-TK-renilla luciferase, as described in Materials and Methods. (A) Twenty-four hours after transfection, 7x10⁴ OP9 cells stably expressing either VSV-Dll1 (dark gray bars, Dll1-OP9), VSV-Dll1AG (black bars, Dll1AG-OP9) or control cells (light gray bars, parental OP9) were added. Luciferase activity was measured after 20 hours of coculture. The relative luciferase activity in the presence of Dll1-OP9 was defined as 100%. Bottom panel shows the expression levels of full-length Dll1 and Dll1AG in the cell lines used for coculture. (B) Increasing amounts of Dll1-OP9 and Dll1AG-OP9 cells were cocultured with Notch1 expressing cells and the Notch reporter activity was measured. Error bars represent the standard variation of triplicate experiments.</p

Cell treatment with smase/coase increases Dll1 turnover.

<p>HeLa cells were transiently-transfected with plasmids encoding Dll1-Apa and GFP. Cells were incubated in the absence or presence of 0.1 unit/mL sphingomyelinase and 1 unit/mL cholesterol oxidase (S/C) and lysed at the indicated time (min) following cycloheximide treatment. Dll1FL, GFP and endogenous transferrin receptor (TfR) were detected by western blotting. A graphic representation of the relative abundance of Dll1, quantified using the Quantity One software (Biorad), is shown in the bottom panel. This result is representative of 3 independent experiments.</p