FIG. 6.

The knockdown of NS does not lead to nucleolar disruption, but its activation of p53 requires the ribosomal proteins L5 and L11. (A) The knockdown of endogenous NS does not disrupt the nucleolus. U2OS cells were transfected with scrambled (Scr) or NS siRNA as indicated. The cells were then stained with anti-B23 (red) and anti-NS (green) antibodies, as well as with DAPI for DNA. (B) The knockdown of endogenous NS enhances the interaction of MDM2 with L5 and L11. U2OS cells were transfected with scrambled or NS siRNA. Cell lysates were subjected to co-IP using anti-MDM2 (4B11) antibodies, followed by IB to detect the level of L11, L5, and MDM2. (C) The knockdown of L5 abolished the induction of p53 by the knockdown of NS. U2OS cells were transfected with scrambled siRNA, with NS siRNA, or with L5 siRNA as indicated. Cell lysates were subjected to IB to detect the expression of p53, MDM2, p21, L5, or NS, as indicated. (D) The knockdown of L11 abolished the induction of p53 by the knockdown of NS. U2OS cells were transfected with scrambled siRNA, with NS siRNA, or with L11 siRNA as indicated. The cell lysates were subjected to IB to detect the expression of p53, MDM2, p21, L11, or NS, as indicated. (E) The knockdown of L5 or L11 abolished the induced expression of the p21 and mdm2 mRNA by the knockdown of NS. U2OS cells were transfected with scrambled siRNA, NS siRNA, L5 siRNA, or L11 siRNA as indicated. Total RNAs were extracted and subjected to RT reactions, followed by real-time PCR assays. Relative expression of the p21 and mdm2 genes was normalized against the expression of GAPDH.