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Figure 5


Fig. 5. Extension of CEF lifespan with SV40 T-antigen and shRNAs. (A) CEFs were infected at PD 37 with a retrovirus encoding SV40 T-antigen (filled squares) or empty vector control (open circles) and passaged until they reached senescence. (B) shRNA-mediated knockdown of INK4b, ARF and p53 in CEFs. For INK4b, the panel shows effects of two separate shRNAs (#2 and #4) as assessed by immunoblotting with the SK14 antibody. Knockdown of ARF expression was assessed by northern blotting of total RNA as described in Fig. 2. GAPDH served as a control for RNA loading. Knockdown of p53 was assessed by immunoblotting with the HP64 antibody. (C) Growth curves of CEFs infected at PD 30 with pRetroSuper encoding independent shRNAs against INK4b (filled squares), a single shRNA against ARF (open triangles) or empty vector (open circles). (D) An analogous experiment showing CEFs infected at PD 40 with pRetroSuper encoding shRNA against p53.





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