Elisabetta Damiani, Nahum Puebla-Osorio, Enrique Gorbea, Stephen E

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Platelet-Activating Factor Induces Epigenetic Modifications in Human Mast Cells Elisabetta Damiani, Nahum Puebla-Osorio, Enrique Gorbea, Stephen E. Ullrich Journal of Investigative Dermatology Volume 135, Issue 12, Pages 3034-3040 (December 2015) DOI: 10.1038/jid.2015.336 Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CXCR4 expression in platelet-activating factor (PAF)-treated HMC-1 cells. (a) Immunoblotting analysis of CXCR4 protein expression 6–24hours after carbamyl-PAF (cPAF; 10μM) treatment. p84 was the loading control. (b) Flow cytometry was used to measure CXCR4 surface expression 24hours post-cPAF treatment. (c) CXCR4 mRNA levels were increased 24hours post-cPAF treatment. Data represent the mean±SEM (N=3). *P<0.05 vs. control (Student’s t-test). Journal of Investigative Dermatology 2015 135, 3034-3040DOI: (10.1038/jid.2015.336) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Platelet-activating factor (PAF) decreases the expression of DNMT1 and 3b. (a) DNMT1 protein expression in carbamyl-PAF (cPAF)-treated HMC-1 cells was analyzed by immunoblotting; p84 is the loading control. (b) Time course of cPAF (10μM)-induced DNMT1 depression. (c) Treating the cells with a PAF receptor antagonist (AMT-491) blocks the cPAF-induced depression of DNMT1. (d) Time course of cPAF-induced depression of DNMT3b expression. (e) Effect of cPAF on DNMT1 and 3b mRNA expression as measured with real-time quantitative polymerase chain reaction (RT–qPCR). Data represent the mean±SEM (N=3). *P<0.05 vs. control (Student’s t-test). Journal of Investigative Dermatology 2015 135, 3034-3040DOI: (10.1038/jid.2015.336) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Platelet-activating factor (PAF) affects the expression of p300 and HDAC2 in HMC-1 cells. (a) Time course expression of p300 after incubation with 10-μM carbamyl-PAF (cPAF). p84 is the loading control. (b) HDAC2 expression was depressed 24hours post-cPAF (10μM) treatment. Data from three independent experiments are shown. Journal of Investigative Dermatology 2015 135, 3034-3040DOI: (10.1038/jid.2015.336) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Effect of platelet-activating factor (PAF) on histone acetylation. (a) Protein levels of total H3 and acetylated H3 in HMC-1 cells 24hours post-carbamyl-PAF (cPAF; 10μM) treatment. p84 is the loading control. (b) Acetylation of the CXCR4 promoter using chromatin immunoprecipitation assay (ChIP) analysis. HMC-1 cells were treated with 10-μM cPAF and harvested 24hours later. Total H3 was used as positive control, and data were normalized against input DNA. Data represent the mean±SEM (N=4). *P<0.05 vs. control (Mann–Whitney U-test,). (c) PAF upregulates the expression of acetyl-H3 mRNA in normal mast cells. Buffy coat–derived mast cells were treated with increasing concentrations of cPAF, and immunoblotting was used to determine the expression of acetyl-H3 (K9/14/18/23/27). Actin is the loading control. Journal of Investigative Dermatology 2015 135, 3034-3040DOI: (10.1038/jid.2015.336) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Blocking acetylation inhibits platelet-activating factor (PAF)-induced cell surface expression of CXCR4. (a) Mast cells treated with carbamyl-PAF (cPAF; red) or cPAF in the presence of curcumin (green) were analyzed using FACS 24hours post-cPAF treatment. The isotype control is shown in blue. (b) cPAF-induced upregulation of p300 is inhibited by curcumin. An aliquot of the same cells analyzed using FACS was removed and lysed and p300 expression determined by immunoblotting. p84 is the loading control. Journal of Investigative Dermatology 2015 135, 3034-3040DOI: (10.1038/jid.2015.336) Copyright © 2015 The Society for Investigative Dermatology, Inc Terms and Conditions