AAD mutation induces premature cellular senescence. ( A ) GFP+ TopBP1 ki/+ cells were sorted 24 hr after transfection and cultured. Images show the cell density and morphology at D1 and D5. Enlargement shows a representative area of TopBP1 ki/− cells from D5. ( B ) SA- β - galactosidase staining of cells 6 days after shRNA transfection shown in blue. ( C ) Quantification of SA-β- galactosidase positive cells from B. The data represent the mean ± SD of at least 500 cells from 2 independent experiments. P value: Student's t -test. ( D ) Semi-quantitative RT-PCR analysis of RNA isolated from D5 cultures from A. The expression level (indicated on top of each sample) was estimated by quantification normalized to the level of GAPDH and then correlated with GFP-shLuc transfected cells. Two independent experiments were performed which showed equivalent results.