Search

Search Results

4 results found. Results per page: [ 20 ][ 40 ][ 60 ][ 80 ][ 100 ][ 200 ][ 300 ]

Sort by: [ Publication Date ][ Score ]

Year of publication: [ 2025 ][ 2024 ][ 2023 ][ 2022 ][ 2021 ][ 2020 ][ 2019 ][ 2018 ][ 2017 ][ 2016 ][ 2015 ][ 2014 ][ 2013 ][ 2012 ][ 2011 ][ 2010 ][ 2009 ][ Any ]

Direction: [ Desc ][ Asc ]

• Research Paper Volume 12, Issue 8 pp 6558-6569

  Epigenetic modulation of macrophage polarization prevents lumbar disc degeneration

  Relevance score: 5.1241193

  Yang Hou, Guodong Shi, Yongfei Guo, Jiangang Shi

  Keywords: lumbar disc degeneration (LDD), aging, macrophage polarization, DNA methyltransferase 1 (DNMT1), transforming growth factor beta 1 (TGFβ1)

  Published in Aging on April 20, 2020

  Show abstract
  Hide abstract

  Inflammation plays an essential role in the development of lumbar disc degeneration (LDD), although the exact effects of macrophage subtypes on LDD remain unclear. Based on previous studies, we hypothesized that M2-polarization of local macrophages and simultaneous suppression of their production of fibrotic transforming growth factor beta 1 (TGFβ1) could inhibit progression of LDD. Thus, we applied an orthotopic injection of adeno-associated virus (AAV) carrying shRNA for DNA Methyltransferase 1 (DNMT1) and/or shRNA for TGFβ1 under a macrophage-specific CD68 promoter to specifically target local macrophages in a mouse model for LDD. We found that shDNMT1 significantly reduced levels of the pro-inflammatory cytokines TNFα, IL-1β and IL-6, significantly increased levels of the anti-inflammatory cytokines IL-4 and IL-10, significantly increased M2 macrophage polarization, significantly reduced cell apoptosis in the disc degeneration zone and significantly reduced LDD-associated pain. The anti-apoptotic and anti-pain effects were further strengthened by co-application of shTGFβ1. Together, these data suggest that M2 polarization of macrophages induced by both epigenetic modulation and suppressed production and release of TGFβ1 from polarized M2 macrophages, may have a demonstrable therapeutic effect on LDD.

  

  Preparation of AAVs that deplete DNMT1 and TGFβ1. (A) Schematic to show the structure of AAVs carrying shRNA for DNMT1 (shDNMT1) or shRNA for TGFβ1 (shTGFβ1) under a macrophage-specific CD68 promoter. The control AAV carried a scramble sequence under the CD68 promoter. The constructs were connected to a GFP reporter by p2A to allow co-expression by one promoter. (B, C) DNMT1 (B) and TGFβ1 (C) levels were examined in 3 mouse macrophage lines, Raw264.7, J774A.1 and IC-21, by RT-qPCR. (D–G) IC-21 was transduced with different AAVs. DNMT1 levels were determined by RT-qPCR (D) and by Western blot (E). TGFβ1 levels were determined by RT-qPCR (F) and by ELISA (G). *p<0.05. NS: non-significant. N=5.

  
  
  

  

  CD68 promoter in AAVs allows specific targeting of macrophages. After LDD was induced in mice, orthotopic injection of these AAVs into LDD-mice was performed. Mice in the scramble group received an orthotopic injection of 100 μl 10¹¹ AAV-pCD68-scramble; mice in the shDNMT1 group received an orthotopic injection of 100 μl 10¹¹ AAV-pCD68-shDNMT1; mice in the shTGFβ1 group received an orthotopic injection of 100 μl 10¹¹ AAV-pCD68-shTGFβ1; mice in the shDNMT1+shTGFβ1 group received an orthotopic injection of 50 μl 0.5X10¹¹ AAV-pCD68-shDNMT1 and 50 μl 0.5X10¹¹ AAV-pCD68-shTGFβ1. Mice were kept for 4 weeks before analysis. (A, B) F4/80 staining and GFP signals are shown by representative images (A) and by quantification of the percentage of GFP+ cells in all F4/80+ cells (B). NS: non-significant. N=7. Scale bars are 20μm.

  
  
  

  

  Effects of AAVs on cytokine production by macrophages. (A) F4/80+ macrophages in the degeneration zone were isolated by flow cytometry, as shown by representative flow charts. (B–G) ELISA for TNFα (B) IL-1β (C) IL-6 (D) IL-4 (E) IL-10 (F) and TGFβ1 (G). *p<0.05. NS: non-significant. N=7.

  
  
  

  

  Effects of AAVs on macrophage polarization. (A, B) Flow cytometry for CD86 and CD206 on F4/80+ cells from the LDD zone, as shown by representative flow charts (A) and by quantification of the ratio of CD206+ M2 macrophages to CD86+ M1 macrophages (B). *p<0.05. NS: non-significant. N=7.

  
  
  

  

  Co-application of shDNMT1 and shTGFβ1 reduces cell apoptosis in LDD tissue. (A, B) Cells isolated from mouse vertebral pulp and annulus fibrosus were dissociated into single cell populations for a flow-cytometry-based apoptosis assay, shown by representative flow charts (A) and by quantification (B). *p<0.05. NS: non-significant. N=7.

  
  
  

  

  Effects of AAVs on spine proteoglycan and collagen II after LDD. (A, B) RT-qPCR for proteoglycan (A) and collagen II (B) in the LDD zone. *p<0.05. NS: non-significant. N=7.

  
  
  

  

  Effects of AAVs on pain assessment after LDD. Von Frey filament test was applied to evaluate the mechanical and thermal pain in 4 consecutive days. (A) Measurement of mechanically induced withdrawal threshold. (B) Measurement of thermally induced withdrawal latency of the paw. *(in yellow) p<0.05: shDNMT1+shTGFβ1 vs scramble. NS: non-significant. N=7.

  
  
  

• Research Paper Volume 11, Issue 8 pp 2343-2351

  Epigenetic control of Foxp3 in intratumoral T-cells regulates growth of hepatocellular carcinoma

  Relevance score: 8.524992

  Qin Liu, Fang Du, Wei Huang, Xiaoyi Ding, Zhongmin Wang, Fuhua Yan, Zhiyuan Wu

  Keywords: hepatocellular carcinoma, Foxp3, DNMT1, methylation

  Published in Aging on April 21, 2019

  Show abstract
  Hide abstract

  Capability of tumor cells to impede immune response are largely associated with their interaction and regulation of CD4+CD25+ forkhead box transcription factor (Foxp)3+ regulatory T (Treg) cells, which suppress cytotoxic T cell-mediated immunity in the tumor microenvironment. Foxp3 level is critical for development and phenotypic maintenance of Treg, and is regulated by transcriptional control and epigenetic modification. Here, we showed that higher percentage of intratumoral Treg cells was positively correlated with lower Foxp3 promoter methylation in hepatocellular carcinoma (HCC), and both of them were associated with higher tumor grade, larger tumors, and poor prognosis of the patients. We used an adeno-associated virus (AAV) carrying either DNA (cytosine-5)-methyltransferase 1 (DNMT1) or shDNMT1 under a CD4 promoter (AAV-pCD4-DNMT1, AAV-pCD4-shDNMT1) to successfully target T-cells and alter the levels of DNMT1. Intratumoral injection of AAV- pCD4-DNMT1 significantly reduced tumor growth in mice, while intratumoral injection of AAV- pCD4-DNMT1 significantly induced tumor growth, compared to injection of control AAV. Finally, the effects of altering DNMT1 levels in T-cells seemed to affect tumor growth through alteration of methylation status of Foxp3 on promoter and CpG regions. Together, these data suggest that epigenetic control of Foxp3 in intratumoral T cells regulates growth of HCC.

  

  More intratumoral Treg cells are detected in high-grade, large HCCs and correlate with poor prognosis. We examined intratumoral Treg cells (CD4+CD25+Foxp3+) in total T-cells (CD4+) in 40 HCC specimens. (A) Illustration of FAC soring of CD4+CD25+Foxp3+ cells. First, CD4+CD25+ cells were isolated (circled gating in the upper panel), and this population was further purified for Foxp3+ cells (circled gating in the lower panel). (B) Percentage of intratumoral Treg cells in specimens with different tumor grades. (C) The median size of these 40 dissected tumors was used as a cutting point to get 20 small-size HCCs and 20 large-size HCCs. The percentage of intratumoral Treg cells in the large-size HCCs and small-size HCCs was compared. (D) The median level of %Treg was used to separate %Treg-high (n=20) from %Treg-low patients (n=20) to compare their overall five-year survival. *p<0.05. N=40.

  
  
  

  

  Lower Foxp3 promoter methylation is detected in intratumoral T-cells from high-grade, large HCCs and correlate with %Treg in T-cells. (A) The relative methylation levels of Foxp3 promoter in intratumoral T-cells in HCC specimens with different tumor grades by MS-PCR. (B) The relative methylation levels of Foxp3 promoter in intratumoral T-cells in small versus large HCCs by MS-PCR. (C) Correlation between the methylation levels of Foxp3 promoter and the percentage of Treg cells in total intratumoral T-cells. (D) The median level of methylation level of Foxp3 promoter was used to separate methylaition-high (n=20) from methylation-low patients (n=20) to compare their overall five-year survival.*p<0.05. N=40.

  
  
  

  

  Specific target and alteration of DNMT1 levels in T-cells. (A) We used an AAV carrying either DNMT1, or scrambled (as a control) or shDNMT1 under a CD4 promoter (AAV-pCD4-DNMT1, AAV-pCD4-scrambled, AAV-pCD4-shDNMT1) to successfully target T-cells and alter the levels of DNMT1. (B) Direct fluorescence for GFP in transduced cells. (C, D) RT-qPCR (C) and Western blot (D) for DNMT1. *p<0.05. N=5. Scale bars are 20 μm.

  
  
  

  

  Epigenetic alteration in intratumoral T-cells affects tumor growth. A mouse HCC cell line Hepa1-6 that expresses luciferase reporter was subcutaneously implantated into C57/BL6 mice to generate detectable tumor. From the second week after transplantation, intratumoral injection with pAAV-pCD4-DNMT1, or pAAV-pCD4-scrambled, or pAAV-pCD4-shDNMT1 was done every week till 8 weeks, when the mice were sacrificed. (A–B) The bioluminescence analysis at sacrifice, shown by representative images (A), and by quantification (B). (C–D) Measurement of dissected tumor mass, shown by representative images (C), and by quantification (D). *p<0.05. N=3.

  
  
  

  

  Promoter and CpG regions are the major sites where methylation is regulated by DNMT1. (A) Intratumoral T-cells were purified by CD4-based flow cytometry from the HCC-bared mice that had received intratumoral injection with AAV-pCD4-DNMT1, or AAV-pCD4-scrambled, or AAV-pCD4-shDNMT1. (B) RT-qPCR for DNMT1 in CD4+ cells from AAV-pCD4-DNMT1-, or AAV-pCD4-scrambled-, or AAV-pCD4-shDNMT1- injected tumor. (C) % CD4+CD25+Foxp3+ Treg cells in total CD4+ cells. (D–F) Methylation status of 3 known sites on Foxp3 (promoter (D), enhancer (E) and CpG (F) region) were assessed by MS-PCR. *p<0.05. NS: non-significant. N=3.

  
  
  

• Research Paper Volume 11, Issue 6 pp 1695-1715

  Long non-coding RNA CDKN2B-AS1 reduces inflammatory response and promotes cholesterol efflux in atherosclerosis by inhibiting ADAM10 expression

  Relevance score: 5.443609

  Haocheng Li, Song Han, Qingfeng Sun, Ye Yao, Shiyong Li, Chao Yuan, Bo Zhang, Bao Jing, Jia Wu, Ye Song, Haiyang Wang

  Keywords: atherosclerosis, long non-coding RNA CDKN2B-AS1, ADAM10, DNMT1, inflammatory response, cholesterol efflux

  Published in Aging on March 29, 2019

  Show abstract
  Hide abstract

  Introduction: Long non-coding RNAs (lncRNAs) play key roles in the development of atherosclerosis through the inflammatory pathway. This study aimed to investigate the role of lncRNA cyclin-dependent kinase inhibitor 2B antisense RNA 1 (CDKN2B-AS1) in atherosclerosis via its function in A disintegrin and metalloprotease 10 (ADAM10).

  Methods: Initially, the expression of CDKN2B-AS1 and ADAM10 in atherosclerotic plaque tissues and THP-1 macrophage-derived foam cells was determined, after which the cholesterol efflux rate of macrophages was calculated. Interaction between CDKN2B-AS1 and ADAM10 was analyzed, after which, expression of CDKN2B-AS1 and ADAM10 were altered to explore their effects on inflammatory response and cholesterol efflux. The aforementioned findings were further intended to be validated by the atherosclerosis mouse model experiments.

  Results: Atherosclerotic plaque tissue and THP-1 macrophage-derived foam cells exhibited downregulated CDKN2B-AS1 and upregulated ADAM10. Upon overexpressing CDKN2B-AS1 or silencing ADAM10, lipid accumulation was reduced and cholesterol efflux was increased. CDKN2B-AS1 located in the nucleus could bind to DNA methyltransferase 1 (DNMT1) to enhance methylation of ADAM10 promoter, leading to suppressed atherosclerotic inflammatory response and promoted cholesterol efflux.

  Conclusion: Altogether, lncRNA CDKN2B-AS1 can inhibit the transcription of ADAM10 via DNMT1-mediated ADAM10 DNA methylation, consequently preventing inflammatory response of atherosclerosis and promoting cholesterol efflux.

  

  CDKN2B-AS1 is downregulated in atherosclerosis. (A) RT-qPCR was used to detect the transcriptional level of CDKN2B-AS1 in atherosclerotic plaque and IMA tissues; n = 16; (B) RT-qPCR determination of transcription level of CDKN2B-AS1 in ox-LDL-exposed THP-1 macrophage-derived foam cells and THP-1 macrophages; * p < 0.05 vs. the IMA tissues or THP-1 cells; the measurement data were expressed in the form of mean ± standard deviation and analyzed by unpaired t-test, the experiment was repeated 3 times; IMA, internal mammary artery; THP-1, the human monocytic leukemia cell line; RT-qPCR, reverse transcription quantitative polymerase chain reaction; CDKN, cell-dependent kinase inhibitor.

  
  
  

  

  CDKN2B-AS1 suppresses atherosclerotic inflammatory response and promotes cholesterol efflux. (A) the infection efficiency of lentiviral vector expressing oe-CDKN2B-AS1; (B) liquid scintillation counter was used to detect the cholesterol efflux of each group; (C) oil red O staining was used to detect the intracellular lipid accumulation of each group (× 200), the red arrows indicate intracellular lipid particles after staining; (D) ELISA was used to detect the levels of IL-1β and TNF-ɑ in each group; * p < 0.05 vs. the oe-NC group; the measurement data were expressed in the form of mean ± standard deviation and analyzed by unpaired t-test, the experiment was repeated 3 times; NC, negative control; ELISA, enzyme linked immunosorbent assay; TNF, tumor necrosis factor; IL, interleukin; CDKN, cell-dependent kinase inhibitor.

  
  
  

  

  ADAM10 is upregulated in atherosclerosis. (A) RT-qPCR detects the transcriptional level of ADAM10 in atherosclerotic plaque and IMA tissues; n = 16; (B) the protein level of ADAM10 in atherosclerotic plaque and IMA tissues determined by Western blot analysis; n = 16; (C) RT-qPCR was used to detect the transcriptional level of ADAM10 in ox-LDL-exposed THP-1 macrophage-derived foam cells and THP-1 macrophages; (D) Western blot analysis was used to detect the protein level of ADAM10 protein in atherosclerotic plaque and IMA tissues;* p < 0.05 vs. the IMA tissues or THP-1 cells; the measurement data were expressed in the form of mean ± standard deviation and analyzed by unpaired t-test, the experiment was repeated 3 times; IMA, internal mammary artery; THP-1, the human monocytic leukemia cell line; ADAM10, A disintegrin and metalloprotease 10; RT-qPCR, reverse transcription quantitative polymerase chain reaction; CDKN, cell-dependent kinase inhibitor.

  
  
  

  

  ADAM10 silencing suppresses inflammatory response and promotes cholesterol efflux in atherosclerosis. (A) the expression of ADAM10 after infection with lentiviral vector expressing sh-ADAM10 determined by RT-qPCR; (B) liquid scintillation counter was used to detect the cholesterol efflux of cells infected with lentiviral vector expressing sh-ADAM10; (C) oil red O staining was used to detect the lipid accumulation of cells infected with lentiviral vector expressing sh-ADAM10 (× 200), the red arrows indicate intracellular lipid particles after staining; (D) ELISA was used to detect IL-1β and TNF-ɑ in cells infected with lentiviral vector expressing sh-ADAM10; * p < 0.05 vs. the sh-NC group; the measurement data were expressed in the form of mean ± standard deviation and analyzed by unpaired t-test, the experiment was repeated 3 times; ELISA, enzyme linked immunosorbent assay; TNF, tumor necrosis factor; IL, interleukin.

  
  
  

  

  CDKN2B-AS1 promotes ADAM10 methylation by recruiting DNMT1. (A) the expression of CDKN2B-AS1 and transcription level of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1 determined by RT-qPCR; (B) Western blot analysis was used to detect the protein band and level of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (C) FISH was used to detect CDKN2B-AS1 localization in cells (× 200); (D) MS-PCR was used to detect the electrophoresis band of ADAM10 methylation level in atherosclerotic plaque, IMA tissues and ox-LDL-exposed THP-1 macrophages or ox-LDL-exposed THP-1 macrophages treated with 5-aza-dc or M.SssI; (E) CHIP assay to detect the output percentage of ADAM10 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (F) RIP assay to detect the output percentage of CDKN2B-AS1 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; (G) RNA pull down to detect the DNMT1 protein pulled down by lncRNA CDKN2B-AS1 in cells infected with lentiviral vector expressing oe-CDKN2B-AS1 or sh-CDKN2B-AS1; In panel D, U represents the un-methylated lane, and M is representative of the methylation lane; * p < 0.05 vs. the oe-NC group; # p < 0.05 vs. the sh-NC group; the measurement data were expressed in the form of mean ± standard deviation and analyzed by one-way ANOVA, the experiment was repeated 3 times; THP-1, the human monocytic leukemia cell line; RT-qPCR, reverse transcription quantitative polymerase chain reaction; CDKN, cell-dependent kinase inhibitor; ANOVA, analysis of variance; ELISA, enzyme linked immunosorbent assay; RIP, RNA-binding protein immunoprecipitation; FISH, fluorescence in situ hybridization; CHIP, chromatin immunoprecipitation; MS-PCR, methylation-specific PCR.

  
  
  

  

  Overexpression of CDKN2B-AS1 methylates ADAM10, inhibits inflammatory response, and promotes cholesterol efflux in atherosclerosis. (A) Oil red O staining of aortic was used to detect aortic plaque formation in C57BL/6J and ApoE-^/- mice (× 10), the red arrows indicate the formed atheromatous plaque; (B) HE staining was used to detect arterial plaque formation in C57BL/6J and ApoE-^/- mice (× 400), the red arrows indicate atheromatous plaque accompanied by foam cells after HE staining; (C) Liquid scintillation counter was applied to measure the effect of various intervention factors on serum cholesterol efflux in ApoE^-/- mice; (D) Oil red O staining of aortic detection of intervention factors of aortic plaque formation on ApoE^-/- mice (× 10), the arrows indicate the formed atheromatous plaque; (E) HE staining of aortic roots for detection of aortic plaque formation in ApoE^-/- mice (× 400). (F) ELISA was used to detect the serum levels of IL-1β and TNF-ɑ in ApoE^-/- mice; * p < 0.05 the M-oe-ADAM10 group versus the M-oe-NC group; # p < 0.05 the M-oe-CDKN2B-AS1 group versus the M-oe-NC group; the measurement data were expressed in the form of mean ± standard deviation and analyzed by unpaired t-test, n = 12; the experiment was repeated 3 times; HE, hematoxylin-eosin; ELISA, enzyme linked immunosorbent assay; TNF, tumor necrosis factor; IL, interleukin; CDKN, cell-dependent kinase inhibitor; ADAM10, A disintegrin and metalloprotease 10.

  
  
  

  

  LncRNA CDKN2B-AS1 inhibits the transcription of ADAM10 via DNMT1-mediated ADAM10 DNA methylation, consequently preventing inflammatory response of atherosclerosis and promoting cholesterol efflux.

  
  
  

• Research Paper Volume 10, Issue 2 pp 212-228

  Ultraviolet A irradiation induces senescence in human dermal fibroblasts by down-regulating DNMT1 via ZEB1

  Relevance score: 7.8738112

  Yuxin Yi, Hongfu Xie, Xiao Xiao, Ben Wang, Rui Du, Yingzi Liu, Zibo Li, Jun Wang, Lunquan Sun, Zhili Deng, Ji Li

  Keywords: UVA, ZEB1, DNMT1, methylation, senescence

  Published in Aging on February 16, 2018

  Show abstract
  Hide abstract

  In this study, we report the role of DNA methyltransferase 1 (DNMT1) in ultraviolet A (UVA)-induced senescence in human dermal fibroblasts (HDFs). We show that DNMT1 expression was significantly reduced during UVA-induced senescence, and this senescence could be alleviated or aggravated by the up- or down-regulation of DNMT1, respectively. Expression of the transcription factor zinc finger E-box binding homeobox 1(ZEB1) also decreased after UVA irradiation, following a UVA-induced increase of intracellular reactive oxygen species (ROS). We show that ZEB1 binds to the DMNT1 promoter and regulates its transcription, which, in turn, affects cellular senescence. These changes in DMNT1 and ZEB1 expression following UVA exposure were confirmed in matched skin specimens that had or had not been sun-exposed. On analyzing the promoter methylation of 24 senescence associated genes in these matched skin specimens, we discovered that p53 promoter methylation was significantly reduced in sun-exposed skin. In vitro experiments confirmed that UVA irradiation reduced p53 promoter methylation, and DNMT1 up-regulation could reverse this effect. Collectively, down-regulation of ZEB1 caused by UVA induced ROS could transcriptionally inhibit DNMT1, leading to low methylation level of senescence related proteins p53 and increase its expression, eventually result in cellar senescence.

  

  DNMT1 attenuates UVA-induced senescence in HDFs. (A)Senescence-associated β-galactosidase (SA-β-gal) activity in HDFs, showing representative images from three independent experiments (a), (scale bar = 200 µm), and the mean percentage of SA-β-gal-positive cells (b).Error bars represent standard deviation from the mean.* vs control, P < 0.05. (B). (a) DNMT1, p53, p21, and p16 mRNA expression, as determined by real-time PCR. Each sample was analyzed in triplicate for each condition. Data are shown as the mean of three independent experiments. * vs control, P < 0.05. (b) DNMT1, p53, p21, and p16 protein expression, as determined by Western blot analysis (left panels). Bar graphs (right panels) show quantitative analysis of scanning densitometric values of these proteins as ratios to β-actin, which was used as a loading control. Data are representative of three independent experiments. * vs control, P < 0.05. (C) DNMT1 expression at the mRNA level (a, b) and the protein level (c, d), determined by real-time PCR or Western blotting, respectively, in HDFs transfected with either DNMT-cDNA or DNMT-shRNA expressing lentivirus (n = 3).* vs DNMT-vector or control-shRNA, P< 0.05. (D)Western blots images (upper panels) and quantitative analysis (lower panels) showing p53, p21, and p16 protein expression. Data are epresentative of three independent experiments. (E)Senescence-associated β-galactosidase(SA-β-gal) activity in cells under the indicated conditions. Representative images are shown (scale bar = 200 µm). The percentages of SA-β-galpositive cells under each condition are presented as the mean ± standard deviation of three independent experiments. * vs DNMT-vector or control-shRNA, P < 0.05;# vs DNMT1-vector+UVA or control-shRNA+UVA, P < 0.05.

  
  
  

  

  ZEB1 attenuates UVA-induced senescence in HDFs via DNMT1. (A) ZEB1 and DNMT1 expression in HDFs at the mRNA (a, b) and protein (c, d) levels following ZEB1 over-expression or knockdown, as determined by real-time PCR and Western blotting, respectively (n = 3).* vs ZEB1-vector or negative control (NC)-siRNA, P< 0.05. (B) Western blots images (upper panels) and quantitative analysis (lower panels), representative of three independent experiments, were showing p53, p21, and p16 protein expression. (C) Senescence-associated β-galactosidase(SA-β-gal) activity of cells under the indicated conditions. Representative images are shown (scale bar = 200 µm). The percentages of SA-β-galpositive cells under each condition are presented as the mean ± standard deviation of three independent experiments. * vs ZEB1-vector or NC-siRNA, P < 0.05;# vs ZEB1-vector+UVA or NC-siRNA+UVA, P < 0.05. (D) Western blots images (left panels) and quantitative analysis (right panels), representative of three independent experiments, showing p53, p21, and p16 protein expression in HDFs co-transfected with ZEB1-cDNA and DNMT1-shRNA. (E) SA-β-gal activity of cells under the indicated conditions, following DNMT1 knockdown. Cells were analyzed as described in (C). * vs ZEB1-vector, P < 0.05;# vs ZEB1-vector+UVA, P < 0.05; $ vs ZEB1-cDNA+UVA.

  
  
  

  

  ZEB1 binds directly to the DNMT1 promoter and regulates its transcription. (A) Schematic showing the region of the DNMT1 promoter containing potential ZEB1 binding sites. (B) Chromatin immunoprecipitation data from HDFs incubated with either anti-ZEB1antibody or non-specific control IgG, showing the amplification of each of the four predicted ZEB1 binding sites within the DNMT1 promoter (termed DNMT1 b1, 2, 3, and 4). Experiments were performed in triplicate.* vs IgG, P < 0.05;ns vs IgG, P>0.05. (C) Luciferase reporter assay data, showing the activity of either the wild type (WT)DNMT1 promoter or mutants lacking each of the predicted ZEB1 binding sites. Cells were transfected with the following plasmids: ZEB1: ZEB1-cDNA-expressing vector; WT DNMT1: reporter plasmid containing WT DNMT1 promoter; DNMT1 Mut1-4: reporter plasmids containing the DNMT1 promoter with putative ZEB1 binding sites 1-4 deleted. Experiments were performed in triplicate. * vs pCWV-ZEB1+pGL4.1, P<0.05; # vs pCWV-ZEB1+pGL4.1-DNMT1, P <0.05.

  
  
  

  

  UVA irradiation regulates transcription factor ZEB1 via ROS. (A) ZEB1 mRNA (a) and protein (b) expression, assessed by real-time PCR and Western blotting, respectively, in irradiated and non-irradiated HDFs. Experiments were performed in triplicate. Intracellular ROS levels were assessed by measuring dichlorofluorescein (DCF) in triplicate experiments. * vs control, P < 0.05. (B) Following treatment with N-acetyl-L-cysteine (NAC), the expression of ZEB1, p53, p21, and p16 was assessed at the protein (a) and mRNA (b) levels. Experiments were performed in triplicate. * vs control or UVA, P < 0.05; Senescence-associated β-galactosidase (SA-β-gal) activity was assessed to evaluate cellular senescence after NAC treatment. In each condition the number of SA-β-gal-positive cells were counted (c, d; scale bar=200 µm). Experiments were performed in triplicate. * vs control or UVA, P < 0.05; Western blots images (left panels) and quantitative analysis (right panels) are representative of three independent experiments.

  
  
  

  

  DNMT1 regulates p53 by modifying CpG methylation. (A) Relative methylation of the promoters of the senescence-associated genes p53, p21, and p16, in sun-exposed or non-sun-exposed human skin samples, * vs non-sun exposed, P < 0.05. (B) Relative methylation of the p53 promoter region in untreated HDFs(a), UVA-irradiated HDFs (b), and UVA-irradiated HDFs over-expressing DNMT1(c). Each horizontal line represents an individual DNA molecule, and the circles represent CpG dinucleotides. Filled circles: methylated CpGs; open circles: unmethylated CpGs. Numbers at the bottom of the figure indicates CpG position.

  
  
  

  

  DNMT1 and ZEB1 expression is reduced in sun-exposed human skin. (A)Representative immunohistochemical staining images of matched human sun-exposed or non-sun-exposed skin specimens from within the same individual. Sections were imaged using primary antibodies raised against DNMT1 or ZEB1 (C, D) (Magnification= 400×). (B) Bar graph shows quantitative analysis of scanning density values of these proteins, * vs non-sun exposed, P < 0.05.