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. 2014 Mar 19;9(3):e91983. doi: 10.1371/journal.pone.0091983

Figure 5. Notch pathway activation with DSL peptide stimulates tumorsphere formation and increased CSC population.

Figure 5

A. Pancreatic cancer cell tumorspheres were cultured in sphere medium containing increasing concentration of DSL peptide as indicated for 5 days. mRNA levels of Hes1 from treated tumorspheres were analyzed by qRT-PCR and normalized to internal control GAPDH (*p<.001 vs. control). B. Tumorspheres cultured as in A. were Western blotted for cleaved-Notch1, Notch1, Hes1 or β-actin. C. Pancreatic cancer cells cultured in sphere medium containing increasing concentration of DSL analyzed for percentage of CSC by FACS analysis of cells stained with DAPI and antibodies to ESA, CD44, and CD24. Percentage of CSC represents ESA+/CD44+/CD24+ cells as a percentage of live DAPI-negative cells (*p<.02 vs. control). D+E. Tumorsphere formation assay performed on pancreatic cancer cells cultured in increasing concentration of DSL peptide. Representative images shown in D. with quantitation of number of tumorspheres per 1000 cells plated shown in E. (*p<.01 vs. control).