1. Histone H3K27 Demethylase JMJD3 in Cooperation with NF-κB Regulates Keratinocyte Wound Healing 
Jungtae Na, Kwanghyun Lee, Wonho Na, Jee-Yoon Shin, Min-Jung Lee, Tae Young Yune, Hae Kwang Lee, Han-Sung Jung, Won Sun Kim, Bong-Gun Ju 
Journal of Investigative Dermatology 
Volume 136, Issue 4, Pages (April 2016)
DOI: /j.jid
Copyright © 2016 The Authors Terms and Conditions

2. Figure 1
Up-regulation of JMJD3 expression in scratch-wounded keratinocytes. (a) Transcripts of JMJD3 and GAPDH were determined in scratch-wounded HaCaT keratinocytes by quantitative polymerase chain reaction. (b) Lysates from scratch-wounded HaCaT keratinocytes were immunoblotted with anti-JMJD3 and anti–β-ACTIN antibodies, respectively. (c) Up-regulation of nuclear JMJD3 in the region of the wound edge, compared to the region far from the wound edge (outward) (>1 mm). HaCaT keratinocytes after scratching were immunostained with anti-JMJD3 antibody. Nuclei were identified using 4',6-diamidino-2-phenylindole staining. Bar = 50 μm. All data represent mean ± standard error of the mean for three independent experiments. Significance values were *P ≤ 0.05 and **P ≤ 0.01.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

3. Figure 2
JMJD3 is required for wound healing in scratch-wounded keratinocytes. (a, e, g) Transcripts of inflammatory, matrix metalloproteinase (MMP), and growth factor genes were determined in scratch-wounded HaCaT keratinocytes by quantitative polymerase chain reaction. (b, f, h) After control (siCTR) or JMJD3 small interfering ribonucleic acid (siRNA) (siJMJD3) transfection, HaCaT keratinocytes were scratched and transcripts of inflammatory, MMP, and growth factor genes were determined by quantitative polymerase chain reaction. (c) After control or JMJD3 siRNA transfection, HaCaT keratinocytes were scratched and photographed. Bar = 400 μm. (d) Transwell assay was performed. All data represent mean ± standard error of the mean for three independent experiments. Significance values were *P ≤ 0.05 and **P ≤ 0.01.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

4. Figure 3
Histone demethylation activity of JMJD3 is required for inflammation and cell migration in scratch-wounded keratinocytes. (a–c) After DMSO control or GSK-J4 treatment, HaCaT keratinocytes were scratched and transcripts of inflammatory, matrix metalloproteinase (MMP), and growth factor genes were determined by quantitative polymerase chain reaction. (d) After DMSO control or GSK-J4 treatment, HaCaT keratinocytes were scratched and photographed. Bar = 400 μm. Quantitative analysis of scratch wound healing assay. (e) Transwell assay was performed in DMSO control or GSK-J4–treated HaCaT keratinocytes. All data represent mean ± standard error of the mean for three independent experiments. Significance values were *P ≤ 0.05 and **P ≤ 0.01.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

5. Figure 4
NF-κB–dependent keratinocyte wound healing. (a) JMJD3 interacts with NF-κB p65 in scratch-wounded HaCaT keratinocytes. (b) Nuclear localization of NF-κB p65 in the region of the wound edge, compared to the region far from the wound edge (outward) (>1 mm). Bar = 50 μm. (c) Increased phosphorylation of IκB in scratch-wounded HaCaT keratinocytes. (d, f, h) Control (siCTR) or NF-κB p65 small interfering ribonucleic acid (siRNA) (sip65)-transfected HaCaT keratinocytes were scratched, and transcripts of target genes were determined by quantitative polymerase chain reaction. (e, g, i) DMSO control or BAY –treated HaCaT keratinocytes were scratched, and transcripts of target genes were determined by quantitative polymerase chain reaction. All data represent mean ± standard error of the mean for three independent experiments. Significance values were *P ≤ 0.05 and **P ≤ MMP, matrix metalloproteinase.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

6. Figure 5
Recruitment of JMJD3 and NF-κB to the inflammatory, matrix metalloproteinase (MMP), and growth factor gene promoters is correlated with demethylation of H3K27me3 in wounded keratinocytes. (a, b, c) Chromatin immunoprecipitation assay was performed in scratch-wounded HaCaT keratinocytes using the anti-JMJD3, anti–NF-κB p65, anti–NF-κB p50, anti-trimethyl H3K27 (H3K27me3), anti-monomethyl H3K27 (H3K27me1), and anti-phosphorylated RNA polymerase II antibodies, respectively. The occupancy of each protein was determined with quantitative polymerase chain reaction at the gene promoter region encompassing the NF-κB binding site. Chromatin immunoprecipitation assay using normal IgG was performed as a negative control. All data represent mean ± standard error of the mean for three independent experiments. Significance values were *P ≤ 0.05 and **P ≤ 0.01.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions

7. Figure 6
Delayed wound healing in JMJD3/NF-κB–depleted mouse skin. (a) Transcripts of JMJD3 and GAPDH were determined in skin wound by quantitative polymerase chain reaction. (b) Lysates from skin wound were immunoblotted with the anti-IκB, anti-phospho IκB, and anti-β-ACTIN antibodies, respectively. (c, d) After application of JMJD3 or NF-κB p65 small interfering ribonucleic acid (siRNA), wound tissue was harvested and transcripts of inflammatory, matrix metalloproteinase (MMP), and growth factor genes were determined by quantitative polymerase chain reaction. Control siRNA was used as a negative control. (e, f) After application of JMJD3 or NF-κB p65 siRNA, the wounded skins were photographed, and percentage of wound area was measured quantitatively. Control siRNA was used as a negative control. All data represent mean ± standard error of the mean for three independent experiments. Significance values were *P ≤ 0.05 and **P ≤ 0.01.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2016 The Authors Terms and Conditions