FIG 4.

SOX9 promotes cancer stem-like properties. (A) Tumor sphere formation in H460 cells expressing the empty vector or each of the two shRNAs against SOX9. The data are for 3 biological replicates. The data are presented as the mean ± SD. **, P < 0.0001 by one-way ANOVA with Tukey’s multiple-comparison test. Bar = 500 μm. (B, C) Same as for panel A but with the empty vector and SOX9-overexpressing cells (B) and secondary tumor spheres formed by the cells from primary spheres (C). Means ± SD are shown. *, P = 0.005 for primary spheres (B) and 0.012 for secondary spheres (C) by unpaired two-tailed t test. The data are for 3 biological replicates. (D) (Left) Representative pictures of tumor spheres formed by A549 cells expressing the empty vector or shRNAs against SOX9 (sh-1 and sh-2) under standard conditions or with cisplatin (1.5 μM) treatment. Bar = 300 μm. (Right) Quantification of spheroids after treatment with cisplatin. **, P = 0.0042 by one-way ANOVA followed by Tukey’s multiple-comparison test. Bars are the mean ± SD (n = 3). (E) As for panel B, plus the cells were exposed to 0.25 μM cisplatin (H460 cells) and 1.0 μM cisplatin (A549 cells) for the duration of the experiment. *, P = 0.043 and P = 0.0106 by unpaired two-tailed t test for H460 and A549 cells, respectively. The data are for 3 biological replicates. (F) Decreased mRNA expression of stem-cell markers in SOX9 knockdown cells (sh-1 and sh-2) versus vector control cells. The data are presented as the mean ± SD. *, P < 0.05 compared with the vector control cells by two-way ANOVA followed by Bonferroni posttests. (G) SOX9 overexpression slows down cell proliferation in H460 and A549 cells. The proliferation rate of the indicated cells was analyzed by counting the cells every 2 days. The data are for 3 biological replicates. ***, P = 0.011 for H460 cells and P = 0.0068 for A549 cells by two-way repeated-measures ANOVA followed by Bonferroni posttests. Means ± SD are shown.