Research Paper Volume 9, Issue 10 pp 2137—2162

Figure 3. The regulation of p53 target genes by SOCS1 is not dependent on a disabled RB pathway. (A) QPCR validation of the p53 target genes identified by microarray analysis but in normal IMR90 fibroblasts expressing either an empty vector (V) or a constitutively activated STAT5A (cS5A) and with either a control shRNA (shNTC) or an shRNA against SOCS1 (shS1). Cells were collected 7 days after infection. (B) SOCS1 knockdown efficiency measured by qPCR in the conditions described in (A). (C) Status of the cells was assessed at the day of RNA collection (day 7 post infection) with a Senescence-Associated β-Galactosidase staining. Positively stained and unstained cells were counted under a light microscope in order to obtain the percentage of senescent cells. (D) Growth curves. Normal human fibroblasts (IMR90) were retrovirally infected with either an empty vector (V) or with constitutively activated STAT5A (cS5A) and with either a control shRNA (shNTC) or a shRNA against SOCS1 (shS1 a). Cells were counted and plated for the growth assay. (E) Western blots of senescence markers (MCM6, pRb and SOCS1) of cells as in (A). Tubulin was used as a loading control. All experiments were performed three times, error bars indicate the standard errors of triplicates, * = p<0.05, using the Student’s t test, **=p<0.01, ***=p<0.005