1. Inhibition of DNA Methylation in the COL1A2 Promoter by Anacardic Acid Prevents UV- Induced Decrease of Type I Procollagen Expression 
Min-Kyoung Kim, Eun Ju Kim, Yao Cheng, Mi Hee Shin, Jang-Hee Oh, Dong Hun Lee, Jin Ho Chung 
Journal of Investigative Dermatology 
Volume 137, Issue 6, Pages (June 2017)
DOI: /j.jid
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2. Figure 1
Anacardic acid (AA) recovered UV-induced decrease of procollagen expression. Human dermal fibroblasts (HDFs) were irradiated with 150 mJ/cm2 of UV or sham irradiation and treated with AA or vehicle for 6 hours. (a) The amounts of procollagen proteins released into culture media from HDFs were analyzed at 24 hours after treatment by Western blotting. Acetylated histone H3 in total cell lysates from acid extraction was analyzed by Western blotting. Bar graphs show densitometric quantitation of procollagen and acetylated histone H3 as ratios to β-actin, which was used as the loading control. (b) The expression of procollagen mRNA was quantified by real-time PCR and was normalized to the respective 36B4 as a control. Values represent the mean ± standard error of the mean of data from three independent experiments. ∗P < 0.05 versus control, ∗∗P < 0.01 versus control, †P < 0.05 versus UV. AA, anacardic acid-treated cells; AcH3, acetylated histone H3; C, control; UV, UV-irradiated cells; UV/AA, UV-irradiated cells incubated with AA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
The recruitment of acetylated H3, p300, and Smad2/3 to the p300 binding site (P2) of the COL1A2 promoter was decreased by UV but increased after anacardic acid (AA) treatment. (a) Diagram of the 5′-flanking region of the COL1A2 promoter shows the p300 binding sites. The numbers indicate nucleotide positions relative to the translation start site. Putative p300 binding sites (P1, P2) in the COL1A2 promoter are shown. Lines denote the two regions of the COL1A2 promoter amplified by specific primers in the chromatin immunoprecipitation analysis. The DNA fragments were immunoprecipitated using (b) acetylated histone H3, (c) p300, and (d) Smad2/3 antibodies. The COL1A2 promoter fragments were detected using PCR. PCR was performed to amplify the regions between -1896/-1720 for P1 (1818/-1805) and -1446/-1320 for P2 (-1406/-1393) of the COL1A2 gene. COL1A2 expression was quantified by real-time PCR, and values were normalized by input DNA and are representative of three separate chromatin immunoprecipitations. Results are expressed as the mean ± standard error of the mean fold induction versus control levels. ∗P < 0.05 versus control, ∗∗P < 0.01 versus control; †P < 0.05 versus UV. AA, AA-treated cells; C, control; IP, immunoprecipitation; UV, UV-irradiated cells; UV/AA, UV-irradiated cells incubated with AA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
UV increased DNA methylation in the COL1A2 promoter. (a) Schematic diagram of the CpG island (from to -1504, from -531 to -292, and from -170 to -62 transcription start site as +1; accession: AF ) located in the COL1A2 promoter and a putative binding site for p300 (from -1406/-1393). Blue lines indicate the region of the COL1A2 promoter amplified by specific primers in chromatin immunoprecipitation (ChIP). Individual CpG sites are indicated as vertical lines. There are five CpG sites (black vertical lines) relative to the putative binding site for p300 (-1406/-1393) (yellow box) in region 2. (b) HDFs were pretreated with 5-AZA-dC for 48 hours or posttreated with AA for 6 hours. After 24 hours of UV irradiation, cells were harvested, and genomic DNA was extracted. Bisulfite conversion and direct pyrosequencing analysis showed DNA methylation rates in five CpG sites in region 2 of the COL1A2 promoter. (c) Representative pyrosequencing results for region 2 of the COL1A2 promoter. The methylation level in the five CpG sites of region 2 in the COL1A2 promoter was markedly increased by UV irradiation but decreased with 5-AZA-dC treatment. Data from three independent experiments are represented as the mean ± standard error of the mean for fold induction versus control levels. ∗P < 0.05, ∗∗P < 0.01 versus control; †P < 0.05, ††P < 0.01 versus UV. (d) Representative pyrosequencing results for the control and AA and 5-AZA-dC treatments with or without UV irradiation. AA, anacardic acid-treated cells; AZA, 5-AZA-2′-deoxycytidine–treated cells; C, control; ChIP, chromatin immunoprecipitation; CpG, 5′-cytosine-phosphate-guanine-3′; HDF, human dermal fibroblast; UV, UV-irradiated cells; UV/AA, UV-irradiated cells incubated with AA; UV/AZA, UV-irradiated cells incubated with 5-AZA-dC.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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5. Figure 4
Anacardic acid (AA) and 5-AZA-dC inhibited DNA methyltransferase (DNMT) activity. (a) Inhibitory effects of AA or 5-AZA-dC were determined by DNMT-1 activity through an indirect ELISA that detects anti-5-methylcytosine antibody. Recombinant DNMT1 was incubated with substrate and inhibitors. DNMT1 activity was measured in the presence and absence of AA or 5-AZA-dC. Data represent the mean ± standard error of the mean from three independent experiments. ∗P < 0.05 versus enzyme control (EC). (b) Quantitative analysis of global DNA methylation. HDFs were pretreated with 5-AZA-dC for 48 hours or posttreated with AA for 6 hours. After 24 hours of UV irradiation, genomic DNA was isolated. Global methylation status determined by specifically measuring levels of 5-methylcytosine (5-mC) was colorimetrically quantified in a microplate-based ELISA assay. Values were derived as percentage of 5-mc compared with control cells and represent the mean ± standard error of the mean. P-values represent treatment values compared with the control. (c) HDFs were pretreated with 5-AZA-dC for 48 hours or posttreated with AA for 6 hours. After 24 hours of UV irradiation, whole-cell lysates were extracted, and DNMT1, DNMT3A, and DNMT3B protein levels were analyzed by Western blot. (d) HDFs were posttreated with AA 6 hours after UV irradiation and harvested after 24 hours. The ChIP assay was performed using the DNMT1 antibody at the P1 and P2 sites of the COL1A2 promoter. ∗P < 0.05 versus control, †P < 0.05 versus UV. 5-AZA, 5-AZA-deoxycytidine–treated cells; 5-AZA-dc, 5-AZA-deoxycytidine; 5-mc, 5-methylcytosine; AA, AA-treated cells; C, control; DNMT, DNA methyltransferase; EC, enzyme control; HDF, human dermal fibroblast; IP, immunoprecipitation; M, mol/L; UV, UV-irradiated cells; UV/5-AZA, UV-irradiated cells incubated with 5-AZA-deoxycytidine.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
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6. Figure 5
ChIP assay for histone H3 acetylation, p300, Smad2/3, and DNMT1 to a P2 site at the COL1A2 promoter in HDFs with pretreatment of 5-AZA-dC. HDFs were UV irradiated and pretreated with 5 μmol/L 5-AZA-dC for 48 hours, and the DNA fragments were immunoprecipitated using (a) acetylated histone H3, (b) p300, (c) Smad2/3, and (d) DNMT1 antibodies. The COL1A2 promoter fragments were detected using PCR. PCR was performed to amplify the regions between -1896/-1720 for P1 and -1446/-1320 for P2 of the COL1A2 gene. The COL1A2 expression was quantified by real-time PCR, and values are normalized by input DNA. Results are presented as the mean ± standard error of the mean fold induction versus control levels. ∗P < 0.05 versus control; †P < 0.05 versus UV. 5-AZA-dc, 5-AZA-deoxycytidine; AcH3, acetylated histone H3; AZA, 5-AZA-dC-treated cells; C, control; ChIP, chromatin immunoprecipitation; DNMT, DNA methyltransferase; HDF, human dermal fibroblast; IP, immunoprecipitation; UV, UV-irradiated cells; UV/AZA, UV-irradiated cells incubated with 5-AZA-dC.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
AA and 5-AZA-dC increased procollagen expression. (a) HDFs were UV-irradiated and pretreated with 5 μmol/L 5-AZA-dC for 48 hours and posttreated with 7.5 μmol/L AA for 6 hours. Procollagen levels in cultured media were determined at 24 hours after UV irradiation by Western blot analysis (n = 3). (b) Schematic diagram of histone and DNA methylation processes in epigenetic regulation of procollagen expression. 5-AZA, 5-AZA-deoxycytidine–treated cells; 5-AZA-dc, 5-AZA-deoxycytidine; AA, anacardic acid-treated cells; AcH3, acetylated histone H3; C, control; DNMT, DNA methyltransferase; HDF, human dermal fibroblast; UV, UV-irradiated cells; UV/5-AZA, UV-irradiated cells incubated with 5-AZA-dC; UV/AA, UV-irradiated cells incubated with AA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions