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. Author manuscript; available in PMC: 2009 Jul 15.
Published in final edited form as: Cancer Res. 2008 Jul 15;68(14):5716–5723. doi: 10.1158/0008-5472.CAN-08-0001

Figure 3. Coculture experiments demonstrate Jagged-1 expressed in feeder cells is essential in promoting cell adhesion and growth of Notch3 expressing cancer cells.

Figure 3

A. Left panel: Western blot analysis shows both Jagged-1 shRNAs, shRNA1 and shRNA2, significantly reduce Jagged-1 protein expression in OVCAR3 cells as compared to the empty vector control. When compared to shRNA1, shRNA2 shows a more potent inhibitory effect. Middle panel: Binding of A2780-GFP cells to Jagged-1 shRNA transfected OVCAR3 cells was examined in a cell-cell association assay. The cell number of Notch3 expressing A2780-GFP cells that adhered to Jagged-1 shRNA-transfected OVCAR3 cells (feeder monolayer) was reduced when compared to the vector-transfected cells at both15 min and 45 min of cocultivation. Right panel: The growth of A2780-GFP cells is significantly reduced when cocultured with Jagged-1 shRNA-transfected feeder cells (OVCAR3) as compared to vector control transfected cells. Data was measured on the 2nd day of coculture. Data also shows similar number of feeder cells (OVCAR3) was present in each experimental group. ** p<0.01; *** p<0.001, Student’s t test. B. Upper panel: Coculture of A2780-GFP cells and J or L feeder monolayer. The cell number of A2780-GFP bound to L cells (without Jagged-1 expression) was lower than that of J cells (with Jagged-1 expression) at both 15 min and 45 min coculture incubation. Lower panel: The growth of A2780-GFP cells is promoted when cocultured with J cells comparing to L cells. Similar number of L and J feeder cells was present in each experiment. ** p<0.01; *** p<0.001, Student’s t test. C. Binding of Jagged-1 to Notch3 was verified by immunoprecipitation/Western experiment. The J cell and A2780-GFP cell coculture lysates were immunoprecipitated with an anti-Notch3 rabbit polyclonal antibody (N3) and were blotted with an HA antibody or a Notch3 antibody. HA antibody was used to detect expression of Jagged-1 in J cells because the expression construct contained an HA epitope tag. Rabbit anti-serum (R) was used as the control in immunoprecipitation. DSG: disuccinimidyl glutarate; DTT: dithiothreitol