p300 Role in Differentiation Figure 5. p21Waf1/CIP1 knockdown inhibits early keratinocyte differentiation of organotypic keratinocyte raft cultures. HFKs were transiently tranfected with p21Waf1/Cip1 targeting siRNA molecules. (A) Immunoblotting of the whole cell lysates with p21Waf’1/CIP1 antibody shows that both siRNA molecules, sip21Waf’1/CIP1A and sip21Waf’1/CIP1B, were able to knockdown p21Waf’1/CIP1 protein levels efficiently (,90%) (B) Western blot shows p21Waf1/CIP1 depletion with sip21Waf1/CIP1 A and a decreased in K1 levels after 48 hours of differentiation. (C) Transfected cells were seeded onto coverslips, incubated with 1.5 mM calcium for 48 hours, and were pulsed with BrdU prior to fixation. BrdU uptake indicates a 90% increased proliferation of p21Waf1/CIP1 knockdown cells prior to the addition differentiation (t = 0). After 48 hours calcium treatment, around 40% of the p21Waf1/CIP1 knock-down cells remained BrdU positive. (D) H&E staining shows an increase in thickness of epithelia in p21Waf1/CIP1 depleted cells (upper panel). Immunohistochemisty staining of organotypic raft cultures indicates that the differentiation marker K1 is reduced in p21Waf1/CIP1 knockdown rafts with low levels of K1 (middle panel). Rafts were pulsed with BrdU for 16 hours prior to harvest and BrdU positive cells counted (lower panel). BrdU positive cells were observed in most parabasal cells. (E) Graph represents BrdU uptake expressed as percentage of scrambled control (Mean +/2 SE, two independent experiments). (F) Stably expressing p300 targeted shRNA cells were transiently transfected with either 1 mg pMT5-p21Waf1/CIP1-Flag vector or pCMV empty vector (negative control) for 24 hours. Transfected cells were then incubated with calcium for 48 hours. Western blot analysis indicates that exogenous expression of p21Waf1/CIP1 rescued the expression of K1 in p300 depleted cells. doi:10.1371/journal.pone.0008369.g005 (Figure 5D & E). Interestingly, BrdU positive cells were also observed in the suprabasal layer of p21WAF1/CIP1 depleted raft, indicating that these cells did not exit cell cycle properly as they migrate upwards. To determine whether exogenous expression of p21Waf1/CIP1 can rescue the proliferation and differentiation defect of p300 depleted cells, we transiently transfected the scramble and shp300 cell lines with either the vector containing human p21Waf1/CIP1 cDNA with a N-terminal Flag tag or pCMV empty PLoS ONE | www.plosone.org 5 January 2010 | Volume 5 | Issue 1 | e8369