p300 Role in Differentiation Knockdown of p300 Increases Life Span of Keratinocytes and Results in Cell Cycle Re-Entry during Differentiation To further study the effects of p300 on cell proliferation we investigated firstly, the effects on proliferation in cycling cells and secondly, if there was an increase in the life span of p300 depleted cells. There was an increase in bromodeoxyuridine (BrdU) uptake in p300 depleted cycling cell (Figure 3A) and also a significant increase in life span, with depleted cells going through senescence after 40 population doublings as opposed to control cells which went though senescence around 25 population doublings (Figure 3B). This result is an average of three experiments using different batches of keratinocytes. Since p300 depletion causes inhibition of differentiation and increased proliferation of cells we reasoned that it may also allow cells to escape from the permanent cell cycle arrest that is required for terminal differentiation of keratinocytes. p300 depleted cells were calcium treated to induce differentiation and after 48 hours the cells were placed in growth medium to determine the capability to re-enter the cell cycle as measured by BrdU uptake. Approximately 25% of depleted cells did not completely exit the cell cycle after 48 hours in calcium while all control cells had exited the cycle (Figure 3C). When after 48 hours in differentiation medium, cells were transferred to growth medium for 24 hours, many p300 depleted cells re-entered the cell cycle compared to control cells (Figure 3C, t = 48+24). To determine if the cells that re-enter the cycle are viable, we replated cells in growth medium and allowed the cells to grow for 7 days. A significant proportion of the p300 depleted cells, but not the control cells, that re-entered the cycle were viable and grew into colonies (Figure 3D). p300 Regulates p21Waf1/CIP1 Promoter Activity during Differentiation To try and understand the mechanism by which p300 depletion causes an increase in proliferation, we determined whether p300 regulation of p21Waf1/CIP1 expression was important. Scramble and shp300 expressing cells were transiently transfected with a pGL-3 plasmid carrying the human p21Waf1/CIP1 promoter and cells were either kept in normal calcium medium (60 mM) or exposed to high calcium concentrations (1.5 mM) for the indicated time points. Luciferase assays showed that the knockdown of p300 down-regulated p21Waf1/CIP1 promoter activity during keratino- cyte differentiation compared to that of the scramble control Figure 3. Stable p300 knockdown increases the proliferation of HFKs and allows the differentiating HFKs to re-enter cell cycle. (A) Cycling HFKs stably expressing p300 targeted shRNAs in culture were pulsed with BrdU prior to fixation. Graph represents percentage of BrdU positive cells, expressed relative to scrambled control in cycling cells (mean +/2 SE of four independent experiments). (B) Graph shows life span progression of stably transduced cell lines. Asterisk (*) indicates the population doubling (PD) level at which senescence occurred. (C) Stably transduced cell lines were seeded onto coverslips and treated with 1.5 mM calcium for 48 hours. After 48 hours, cells were incubated with fresh media for 24 hours (t = 48+24). (D) Differentiated HFKs were replated onto six well plates and cells were stained with crystal violet after incubating with fresh media for 7 days. Histogram represents three independent experiments calculating the average A540 nm reading of crystal violet solutions following destaining of colonies. doi:10.1371/journal.pone.0008369.g003 PLoS ONE | www.plosone.org 3 January 2010 | Volume 5 | Issue 1 | e8369