of p53, and causes decreased expression of p21Waf1/CIP1 and K1 in p300 depleted differentiating HFKs. (C) H&E staining shows an increase in thickness of epithelia in p300 depleted cells (upper panel). Immunohistochemisty staining of organotypic raft cultures indicates that the differentiation markers K1 is reduced in p300 knock-down rafts only (middle panel). Rafts were pulsed with BrdU for 16 hours prior to harvest and BrdU positive cells counted (lower panel). BrdU immunostaining reveals that there is an increase in the number of proliferative cells in the basal layer of p300 knock down rafts compared to control or CBP (lower panel). (D) Graph represents BrdU uptake expressed as percentage of scrambled control (mean +/2 SE, two independent experiments). Found at: doi:10.1371/journal.pone.0008369.s002 (0.49 MB TIF) Figure S3 The p53 response element of p21Waf1/CIP1 promoter is required for its activity in differentiation. (A) Schematic representing reporter vectors used in panels B and C. For the p21Waf1/CIP1-mut1 reporter vector, the distal p53 binding site (RE1) was altered by site-direct mutagenesis to GAAAC. (B) HFKs cells were transfected with constructs shown in panel A. Loss of distal p53 response element inhibits the activation of p21Waf1/CIP1 promoter in differentiating cells. (C) HFKs were transfected with a mutated p21Waf1/CIP1 promoter luciferase reporter vector (p21Waf1/CIP1-mut1). Mutation of the distal p53 response element (RE1) abrogates the induction of p21Waf1/CIP1 promoter activity during differentiation. All luciferase assays were normalized for References 1. Kasten MM, Giordano A (1998) pRb and the cdks in apoptosis and the cell cycle. Cell Death Differ 5: 132–140. 2. Stiegler P, Kasten M, Giordano A (1998) The RB family of cell cycle regulatory factors. J Cell Biochem Suppl 30–31: 30–36. 3. 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