Figure 2. Sox2 depletion decreases transformation properties and tumorigenesis.
(A) Sox2 protein expression in osteosarcoma cells stably expressing Sox2 shRNA. Western blot of mOS-482 osteosarcoma cells expressing scrambled (sc) shRNA or shRNAs against Sox2 were analyzed by immunoblotting. Tubulin was used as a loading control.
(B) Colony formation. Osteosarcoma cells infected with either scrambled (sc) or Sox2 shRNA viruses were selected in Hygromycin B for 7-10 days. Colonies were counted following staining with Crystal Violet. Representative plates of mOS-482 cells are shown. Table shows genotypes and colony counts from four independent cell lines.
(C) Soft Agar Colony Formation; (D), Migration and (E) Invasion. mOS-482 cells expressing sc or Sox2 shRNA were analyzed in the indicated assays as described in Materials and Methods. All experiments were performed in at least two independent cell lines. Results shown are an average of triplicate plates. * = p<0.05
(F) Tumorigenicity assay: Tumor-forming ability of osteosarcoma cell lines (mOS-482) expressing either scrambled or Sox2 shRNAs, was measured in NOD/SCID mice. Injection of 1 × 106 cells gave similar results (not shown). Tumor volume was monitored and measured bi-weekly. Difference between groups was measured by ANOVA. * = p<0.05
The inset shows a western blot of Sox2 protein expression in the injected cells and isolated tumors from either scrambled or long latency tumors arising from cells expressing Sox2 shRNAs.