Thermal Cycler (Bio-Rad) equipped with a Chromo4 Real-Time PCR Detection System (Bio-Rad) and melting curve analysis also performed. In brief cDNA samples were diluted 1:10 and quantified by amplification against serial dilutions of appropriate control cDNA with the following cycling conditions: initial denaturation 95uC for 10 minutes, 40 cycles of 95uC-15 seconds, 58uC-15 seconds, 60uC-60 seconds. Expression levels were assessed in triplicate, normalized to large ribosomal protein (RPLPO) levels and graphs represents the combined results of two independent biological replicates. The specific primers for this analysis were as follows: p21Waf1/CIP1 59 AGCGGAACAAGGAGTCAG 39 (sense) and 59 CGTTAGTGCCAGGAAAGAC 39 (antisense); p53 59 CCAGGGCAGCTACGGTTTC93 (sense) and 59 CTCCGTCA- TGTGCTGTGACTG93 (antisense) [37]. Chromatin Immunopreciptation (ChIP) Assays ChIP assays were carried out using ,26106 keratinocytes fixed with 1.5% formaldehyde for 20 minutes. Cells were washed twice in ice cold PBS and suspended in collection buffer (100 mM Tris- HCl pH 9.4, DTT 10 mM Protease inhibitors). They were then subsequently washed with 1 mL of NCP1 buffer (10 mM EDTA, 0.5 mM EGTA, 10 mM Hepes pH 6.5, 0.25% Triton X-100) and NCP2 buffer (1 mM EDTA, 0.5 mM EGTA, 10 mM Hepes pH 6.5, 200 mM NaCl) to isolate nuclei. Cells were then spun down and resuspended in 1 mL sodium dodecyl sulfate lysis buffer (0.5% Empigen BB, 1% sodium dodecyl sulfate, 10 mM EDTA, 50 mM Tris-HCL pH 8.0) supplemented with complete protease inhibitor cocktail (Roche), and DNA in the cross-linked prepara- tions was sonicated 6 times for 20 seconds each, to an average fragment size of 500 bp using Sonics Virba Cells Sonicator (24% amplitude) (Sonics & Materials Inc. USA). The insoluble material was removed by centrifugation, and soluble chromatin samples were incubated overnight at 4uC with 2 mg of monoclonal antibodies against p53 (clone DO-1; Santa Cruz Biotechnology), p300 (clone C20; Santa Cruz Biotechnology) or control immuno- globulin G (IgG) and 25 ml of pre-blocked Dynabeads Protein G (Invitrogen), made up to 1 ml final volume with IP buffer (1% Triton X-100, 0.1% sodium deoxycholate, 10 mM EDTA, 50 mM Tris-HCL pH 8.0). The next day, the immune complexes were washed eight times with 1 mL of RIPA buffer (50 mM Hepes pH 8.0, 1 mM EDTA pH 8.0, 1% NP40, 0.7% Deoxycholate, 0.5 M LiCl, Protease inhibitor). Afterwards, the complexes were resuspended in 1 mL of 1X TE buffer and spun down at 3000 rpm for 3 minutes. The samples were then eluted with 50 ml of elution buffer (10 mM Tris pH 8.0, 1 mM EDTA, 10% SDS) at 65uC for 10 minutes vortexing every two minutes during incubation. The eluted samples were spun for 30 second at 15000 rpm and then transferred to fresh tubes. The elution process was repeated and 70 ul elution buffer added. Input templates were purified from 5% of the original lysates in parallel with the eluted immunoprecip- itated samples. Cross-linking was reversed by incubating the samples at 65uC for 16 hours. The samples were then purified by using a Qiagen DNA purification kit following the manufacturer’s protocol. The recovered DNA (4 ml from 30 ml immunoprecipi- tated chromatin DNA or 1 ml from the 60 ml input DNA control) was subjected to PCR amplification using GoTaq green Felix PCR reaction kit. The specific primers for this analysis were used as previously described [25,38,39]. Replicative Life Span Determination Cells were plated at 26105 cells per p60 dish in Epilife media, refed every 2 days, and subcultured 3 days after plating, before growth was slowed by high cell density. Population doublings (PD) per passage was calculated as log2(number of cells at time of p300 Role in Differentiation subculture/number of cells plated). Cumulative PD was plotted against total time in culture to determine replicative life span and the onset of senescence. Western Blot Analysis Protein lysate concentrations were 50 mg for western blots as previously described [40]. The following primary antibodies were used in this study: mouse monoclonal anti-p53 (DO-1 Santa Cruz Biotechnology 1:1000), mouse monoclonal anti-p21Waf1/Cip1 (BD Phamingen 1:1000), mouse monoclonal anti-p300 (1B1 Abnova 1:500), rabbit polyclonal anti-acetylated p53 (lysine 382, Cell signaling 1:1000), rabbit polyclonal anti-keratin 1 (Convance 1:2500), mouse monoclonal anti-Flag (Sigma Aldrich, 1::2000) and mouse monoclonal anti-b-Actin (Sigma 1:10000). Luminescence was determined by incubation with Chemiluminescence Detection Reagent (PerkinElmer, USA) and exposed using FluorChem SP imaging system (Alpha Innotech, USA). Immunofluorescent Staining Tissue embedding and haematoxylin and eosin staining were performed by standard techniques. Images were taken on an Olympus BH-2 microscope with an Olympus D25 camera using Cell B software (Olympus). Five micrometer thick paraffin- embedded sections were deparaffinized with xylene, rehydrated with a series of step down concentration of ethanol and antigen retrieval carried out with boiling 1x antigen retrieval buffer (BioGenex, San Ramon, CA). Briefly, sections were permeabilised with 10%FCS/0.2% Trition X-100 for 30 minutes at room temperature, rinsed and incubated with primary antibody overnight at 4 in 10% FCS. The next day, the sections were washed and incubated with secondary antibody at room temperature for 1 hour, washed and mounted with Prolong Gold antifade reagent plus DAPI (Molecular Probes). The following antibodies were used in this study: mouse monoclonal BrdU (BD Pharmingen 1:100), mouse monoclonal anti-filaggrin (Neomarkers 1:50 no retrieval), rabbit polyclonal anti-ki67 (Santa Cruz Biotechnology 1:100), rabbit polyclonal anti-keratin 1 (Covance 1:2500 no retrieval), and goat anti-mouse and anti-rabbit secondary conjugated to Alexafluor 488 nm or 594 nm (Molecular Probes 1:400). BrdU pulsed cells on coverslips were fixed for 15 minutes with 4% formaldehyde, washed 3x with PBS, submitted to antigen retrieval and stained as described above. For raft sections, the number of Ki67 or BrdU positive cells was counted for a minimum of 10 fields of view. For coverslips, a minimum of 10 fields of view and 500 cells were counted on each slide and expressed as percentage of BrdU positive cells (BrdU/ DAPI). In all cases, immunofluorescent images were captured on a Leica AF6000 inverted fluorescence microscope and Leica AF imaging software. Exposure times were kept constant within an experiment. Supporting Information Figure S1 Depletion of p300 does not affect the p53 expression at transcriptional level during keratinocyte differentiation. Stably expressing p300 targeted shRNAs cells were incubated with calcium for the indicated times courses. Real time quantification indicates that p300 knock-down does not affect the expression of p53 at transcriptional level during keratinocyte differentiation. Found at: doi:10.1371/journal.pone.0008369.s001 (0.04 MB TIF) Figure S2 p300 knock-down, but not CBP, inhibits early keratinocyte differentiation of organotypic raft. HFKs were transiently tranfected with either p300 or CBP targeting siRNA molecules. (A and B) Western blots indicate that depletion of p300, but not CBP, reduces the p300 mediated acetylation and expression PLoS ONE | www.plosone.org 7 January 2010 | Volume 5 | Issue 1 | e8369