Research Paper Volume 9, Issue 10 pp 2137—2162

Figure 4. SOCS1 overexpression is sufficient to regulate the expression of SOCS1-dependent p53 target genes. (A) Western blots of SOCS1 and phospho-p53 (p-p53 S15) in U2OS cells expressing either empty vector (V) or SOCS1 (S1). (B) QPCR for p53 target genes in cells as in (A). Cells were collected at day 5 or 7 post-infection. (C) Growth curves of IMR90 cells expressing either empty vector (V) or SOCS1 (S1). (D) Western blots of SOCS1 and phospho-p53 (p-p53 S15) in IMR90 cells expressing either empty vector (V) or SOCS1 (S1). (E) QPCR for p53 target genes in cells as in (C). Cells were collected at day-7 post infection. (F) Senescence associated β-galactosidase of IMR90 cells expressing either empty vector (V) or SOCS1 (S1). Cells were fixed and stained at day 12 post- infection. (G) QPCR of IMR90 cells expressing either a control shRNA (shNTC) or a shRNA against p53 (shp53) combined with SOCS1 (S1) or empty vector (V) to confirm that the genes in (E) are targets of p53. (H) Western blots for the indicated proteins in IMR90 cells expressing a control shRNA (NTC) or an shRNA against p53 (shp53) and also infected with a SOCS1 expressing vector (S1) or a vector control (V). (I) 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. 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.