SW1116 cells (34%) co-expressed CD133 and CD29, suggesting that the in vitro propagated spheroid cells can express both markers (Figure 2A and B). Figure 2 Flow cytometry analysis showing the expressions of CD133 and CD29 (A) and the positive rates of CD133 and CD29 (B) in SW1116 cells. Side population cells in SW1116 cells After concerning cultured for 30 d, SW1116csc and SW1116 cells were stained with Hoechst 33 342 to analyze differences in the SP proportion. The data showed that SW1116csc contained 38.9% �� 7.5% of Hoechst 33342-stained dull cells, while SW1116 cells contained only 1.2% �� 0.3% of Hoechst 33 342-stained dull cells. Telomerase activity of SW1116 cells Telomerase reactivation is essential for stabilization of telomere length in attaining cellular immortality and telomerase is activated in human CSC.

In this study, the RTA of SW1116csc and SW1116 cells was 3 674 �� 287 and 2 518 �� 140, respectively, indicating that the telomerase activity of SW1116csc is higher than that of SW1116 cells (Figure (Figure3,3, P < 0.01). Figure 3 Telomerase activities in SW1116csc and SW1116 cells. RTA: Relative telomerase activities. Expressions of stem cell genes and proteins in SW1116 cells RT-PCR showed that the expressions of CD133, CD29, Musashi-1, ABCG2, TERT genes increased significantly in SW1116 cells, while no change occurred in expressions of Oct-4 and Sca-1 gene in SW1116csc and SW1116 cells (Figure (Figure4A).4A). Western blot showed that the expressions of CD133, CD29, Musashi-1, ABCG2, and TERT proteins in SW1116csc and SW1116 cells were increased at transcriptional level (Figure (Figure4B4B).

Figure 4 Expressions of CD133, CD29, Musashi-1, TERT, ABCG2, Oct-4 and Sca-1 genes (A) and CD133, CD29, Musashi-1, ABCG2 and TERT proteins (B) in SW1116csc and SW1116 cells (left: SW1116 cells, right: SW1116csc). GAPDH: Glyceraldehyde-3-phosphate dehydrogenase; … Proteome differential expression in SW1116csc and SW1116 cells The silver-stained 2-DE gels of proteomes expressed in SW1116csc and SW1116cells were presented. The protein spots detected in gels of total protein were 2 115 �� 137 and 2 133 �� 153, respectively. One of the gels was selected as a reference gel. Student��s t-test showed that the volume of 26 protein spots was significantly changed in gels (Figure (Figure5A,5A, P < 0.05). The expression levels were increased and decreased, Anacetrapib respectively, in 15 and 11 out of the 26 protein spots of SW1116 cells (Figure (Figure5B5B). Figure 5 Two-dimensional gel electrophoresis profile (A) and histogram (B) showing expression levels of 26 protein spots in SW1116 cells and SW1116csc.