FIG. 4.

Reduction of cyclin A₁ protein levels reduces tumorgenicity. (A) Soft-agar tumorgenicity assay. Reduction of cyclin A₁ protein levels reduces tumorgenicity of iPS cells. Quantification and photos of the soft-agar assay, demonstrating that iPS cells treated with stable lentiviral shRNA for cyclin A₁ have reduced number of colonies forming in anchorage-independent conditions compared to controls. Note that the size of the mESC colony in the photos was the smallest assessed in the quantification. Three iPS clones were tested. This suggests that reduction of cyclin A₁ in iPS reduces tumorigenicity. (B) In vivo tumorgenicity assay. Graph showing differences in the size of teratomas formed by iPS cells treated with stable lentiviral shRNA for cyclin A₁ or controls compared to mESCs. (C) Photographs of teratomas formed by mESCs or iPS cells treated with stable lentiviral shRNA for cyclin A₁ or controls stained for Oct4 expression with DAB compared to mESCs. Graph demonstrates quantification of the percent of Oct4-positive cells in teratomas. (D) Heat maps from Agilent gene expression arrays for 3 gene groups for mESCs and miPS cells with and without cyclin A₁ RNAi treatment demonstrating a downregulation of important oncogenes (Myb, Myc, and cyclin D₁) and an upregulation of important tumor suppressor genes (Rb) and pluripotency factors (BMP4), explaining the reduced tumorigenicity and increased pluripotency of iPS cells with reduced cyclin A₁. Color images available online at www.liebertonline.com/scd