A previous report indicated that p/CAF directly binds to Smad3. As we have shown that TGFb induces complex formation between Smad3 and p21, we investigated whether selleck chem Nilotinib endogenous p/CAF is also required for Smad3 association with p21. For this, HEK293 cells were co transfected with myc Smad2, myc Smad3 and flag p21 with or without p/CAF siRNA to block expression of endogenous p/CAF. As shown in Figure 8B, TGFb induced complex formation between p21 and Smad3, independently of Smad2. Interestingly, depletion of p/CAF completely prevented this interac tion, indicating that endogenous p/CAF is required for Smad3 interaction with p21. To investigate whether p/CAF is necessary for the regu lation of p21 dependent TGFb downstream target genes, SUM159 cells were transiently transfected with flag tagged p21 in the presence or the absence of two different p/CAF siRNAs.

The gene expression of p/CAF acetyltransferase 2B, KAT2B was measured to verify the efficiency of p/CAF knockdown by q PCR. Overexpression of p21 potentiated induction of IL6, IL8 and PTGS2 mRNA by TGFb. However, these effects were significantly blocked when p/CAF gene expression was silenced, indicating that p/CAF is required for p21 dependent gene expression of the TGFb targets. The requirement of p/CAF downstream of TGFb was further investigated using the Transwell Matri gel assay. As shown in Figure 8E, knocking down p/CAF gene expression significantly impaired TGFb induced cell invasion. Efficiency of the siRNA was verified by Western blotting.

Because acetyltransferase p/CAF regulates gene tran scription by acetylating histones and transcription factors, we then assessed whether TGFb could induce global changes in histone acetylation in breast cancer cells. For this, total histone proteins were extracted from SCP2 cells, treated or not with TGFb and subjected to immunoblot ting using an acetylated lysine antibody. As shown in Fig ure S8, TGFb had no effect on global histone acetylation while TSA, a histone deacetylase inhi bitor, showed a marked increase in the acetylation levels. This suggested that the functional relevance of the p/CAF recruitment to the p21/Smad complex may be more directed towards acetylation of specific targets rather than Drug_discovery global histone modifications. To address this, we examined whether p/CAF could acetylate p21 and/or the Smads. Interestingly, we found that p/CAF is capable of interact ing with Smad2 and Smad3, leading to an increased acety lation of both Smad proteins. Moreover, the acetylation is specific to Smad2 and Smad3, as p21 did not show any increased acetylation by p/CAF. Smad3 acetylation has been suggested to be required for its DNA binding activity.