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Figure 4


Fig. 4. ANKRD11 regulates p53-mediated expression of p21waf1 in breast cell lines. (A) ANKRD11 expression in the indicated breast cell lines was determined by real-time RT-PCR. Cultures of MCF-7, MB-468 or MB-231 stably expressing GFP-ANKRD11 (MCF-7-ANK-1 to ANK-3; MB-468-ANK-1; MB-231-ANK-1 to ANK-3) or negative control cell lines (MCF-7; MB-468; MB-231) were established from single colonies after retroviral transduction and selected in geneticin. Total ANKRD11 expression (endogenous plus exogenous; light shading) in these stably expressing cell lines were determined and compared with the levels of endogenous ANKRD11 expression (dark shading) in the indicated breast cell lines. The p53 status and 16q LOH status of each breast cell line is indicated. (B) Restoration of ANKRD11 expression increases p21waf1 expression only in MCF-7 and MB-468 cell lines. Both CDKN1A mRNA and p21waf1 protein levels (inset) were determined in cultures stably expressing GFP-ANKRD11 or the negative control cell cultures as described in A using real-time RT-PCR analysis and western blot analysis. (C) ANKRD11 upregulates p21waf1 expression in a p53-dependent manner. MCF-7 or MCF-7-ANK-1 cells as described in A were transfected with either p53-specific or scrambled siRNA for 72 hours. Both p53 and p21waf1 protein levels were determined using western blot analysis, and p21waf1 levels were quantified by densitometry (representative of two independent experiments). (D) Silencing of ANKRD11 by shRNA reduces p53-mediated transcription. MCF-10A cultures stably expressing ANKRD11 shRNA (MCF-10A-shANK) show a reduction in both mRNA and protein levels of ANKRD11 compared with MCF-10A cells stably expressing scrambled shRNA (MCF-10A-SCR). MCF-10A-shANK and MCF-10A-SCR cells were either untreated or treated with mitomycin C for 12 hours and the relative CDKN1A mRNA levels and p53 protein levels were determined using real-time RT-PCR analysis and western blot analysis, respectively.





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