p300 Role in Differentiation Figure 1. Stable p300 depletion inhibits differentiation. HFKs were induced to differentiate in CaCl2 medium and harvested at the indicated time points (t = 0, 6, 16, 48 hrs). (A) Western blot analysis of levels of p300 in control and p300 depleted HFKs using scrambled shRNA (scr) two shRNA molecules (shp300A and shp300B) directed against p300. (B) Cells stably expressing p300 targeting shRNAs were induced to differentiate and harvested at the indicated time points. Western blot analysis indicates that knocking down p300 dramatically reduces the p300 mediated acetylation and expression of p53. There is also a decrease in p21Waf1/CIP1 and K1 levels in p300 depleted HFKs. The results shown are representative of three independent experiments. doi:10.1371/journal.pone.0008369.g001 shRNA molecules directed against p300 and a scrambled control. Western blot analysis indicated that levels of p300 were decreased significantly in both p300 knockdown lines (,80%) (Figure 1A). Scramble and shp300 cell lines were then treated with calcium to induce differentiation and harvested at indicated time points. In control cells p300 dependent acetylation of p53 on K382 was observed, but this is absent in p300 depleted cells due to the significant reduction in p53 protein levels (Figure 1A). These experiments were carried out 3 times with different batches of HFKs and we always observed a decrease in p53 protein levels in p300 depleted cells, although mRNA levels were unchanged compared to control cells (Figure S1). Depletion of p300 also reduces the expression of early differentiation marker, keratin 1 (K1) and p21Waf1/CIP1 during calcium treatment (Figure 1B). We have only shown results with one of the shRNA molecules as the outcome with the other was similar. Reduced levels of induction of differentiation markers was also observed in organotypic raft cultures, where knockdown of p300 resulted in reduced expression of K1 (early marker) and filaggrin (late marker) (Figure 2A). There was also an increase in proliferation of HFKs in the basal layer of the raft cultures in the knockdown cells (Figure 2A and B). Moreover, we transiently knocked down the expression of p300 or CBP with short interfering RNA (siRNA) molecules and induced the cells to undergo differentiation. Strikingly, depletion of p300, but not CBP, down-regulated the p300 dependent p53 protein levels, which in turn reduced the expression of p21Waf1/CIP1 and K1 in differentiating keratinocytes (Figure S2). Since p300 depleted cells expressed normal levels of CBP in differentiation, these results suggest that p300, but not CBP, plays a role in keratinocyte differentiation. Figure 2. p300 has a role in proliferation and differentiation of organotypic keratinocyte raft cultures. (A) H&E staining of organotypic raft cultures of control (scr) and p300 knock down (shp300A) cells (upper panel). Immunostaining of raft sections indicates that level of early marker keratin 1 (K1) and late marker filaggrin (Fil) are significantly reduced in knockdown cells (middle panel). Ki67 immunostaining reveals that there is an increase in the number of proliferative cells in the basal layer of p300 knockdown rafts compared to control (lower panel). (B) Graph represents percentage of Ki67 positive cells in p300 depleted cells, expressed relative to scrambled control (Mean +/2 SE, two independent biological replicates). doi:10.1371/journal.pone.0008369.g002 PLoS ONE | www.plosone.org 2 January 2010 | Volume 5 | Issue 1 | e8369