1. SIRT1, a Class III Histone Deacetylase, Regulates LPS-Induced Inflammation in Human Keratinocytes and Mediates the Anti-Inflammatory Effects of Hinokitiol 
Ju-Hee Lee, Ji-Hong Moon, You-Jin Lee, Sang-Youel Park 
Journal of Investigative Dermatology 
Volume 137, Issue 6, Pages (June 2017)
DOI: /j.jid
Copyright © 2017 The Authors Terms and Conditions

2. Figure 1
Effect of hinokitiol against LPS-induced inflammatory responses in NHEKs. NHEKs were pretreated with hinokitiol and exposed to LPS (100 ng/ml) for 12 hours. Total RNA was extracted to quantify the mRNA expression of (a) TNF-α and (b) IL-6. The (c) TNF-α, (d) IL-6, and (e) PGE2 secreted to media were determined by ELISA. Nuclear/cytosolic proteins were detected by (f) Western blot using anti-NF-κB. (g, h) Scratch assays were photographed under light microscope. Original magnification ×200. (i) The MMP-9 activities were examined by gelatin zymography. Bar graph was generated using mean ± standard error of the mean (n = 3). ∗P < 0.05, ∗∗P < 0.01 indicates significant difference between control and treatment group; #P < 0.05 indicates significant difference compared with LPS treatment group. LPS, lipopolysaccharide; M, mol/L; MMP, matrix metalloproteinase; NHEK, normal human epidermal keratinocyte; PGE2, prostaglandin E2; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

3. Figure 2
Hinokitiol increases Sirt1 expression and activation in NHEKs. In the presence of sirtinol (Sirt1 inhibitor), NHEKs were treated with hinokitiol for 24 hours. (a, c) Protein expression of Sirt1 and acetylation of p53 were determined by Western blot analysis. (b, d) Sirt1 deacetylase activities in nuclear fractions of NHEK cells were analyzed. Bar graph was generated using mean ± standard error of the mean (n = 3). ∗P < 0.05, ∗∗P < 0.01 indicates significant difference compared with control; #P < 0.05 indicates significant difference compared with LPS treatment group. M, mol/L; NHEK, normal human epidermal keratinocyte.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

4. Figure 3
Sirt1 inhibition blocked hinokitiol-induced anti-inflammatory effects in NHEKs. In the presence of sirtinol, NHEKs were pretreated with hinokitiol and exposed to LPS for 12 hours. (a, b) Total RNA was extracted for the quantification of TNF-α and IL-6 mRNA expression levels. The (c) TNF-α, (d) IL-6, and (e) PGE2 secreted to media were determined by ELISA. (f) Nuclear/cytosolic proteins were detected by Western blot using anti-NF-κB. (g, h) Scratch assays in NHEK cells were photographed under light microscope. Original magnification ×200. (i) The MMP-9 activities were examined by gelatin zymography. Bar graph was generated using mean ± standard error of the mean (n = 3). ∗P < 0.05, ∗∗P < 0.01 indicates significant difference compared with control; #P < 0.05 indicates significant difference compared with LPS treatment group. LPS, lipopolysaccharide; MMP, matrix metalloproteinase; NHEK, normal human epidermal keratinocyte; PGE2, prostaglandin E2; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

5. Figure 4
Hinokitiol regulates the Sirt1 expression and activation in NHEKs. After Sirt1 or negative control siRNA transfection, NHEKs were treated with hinokitiol for 24 hours. (a) Protein expression of Sirt1 and acetylation of p53 were determined by Western blot analysis. (b) β-actin was used as loading control. (c) Sirt1 expression was analyzed by immunocytochemistry. Scale bar = 25 μm. Sirt1 deacetylase activities in nuclear fractions of NHEK cells were analyzed. Bar graph was generated using mean ± standard error of the mean (n = 3). ∗P < 0.05 indicates significant difference compared with control; #P < 0.05 indicates significant difference compared with LPS treatment group. LPS, lipopolysaccharide; MMP, matrix metalloproteinase; NC, normal control; NHEK, normal human epidermal keratinocyte; PGE2, prostaglandin E2; siRNA, small interfering RNA; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

6. Figure 5
Sirt1 siRNA decreased hinokitiol-mediated anti-inflammatory effects in NHEKs. After Sirt1 or negative control siRNA transfection, NHEKs were pretreated with hinokitiol in the presence of LPS treatment. Total RNA was extracted to quantify the mRNA expression of (a) TNF-α and (b) IL-6. The (c) TNF-α, (d) IL-6, and (e) PGE2 secreted to media were determined by ELISA. (f) Nuclear and cytosolic proteins were detected by Western blot using anti-NF-κB. (g, h) Scratch assays were photographed under light microscope. Original magnification ×200. (i) The MMP-9 activities were examined by gelatin zymography. Bar graph was generated using mean ± standard error of the mean (n = 3). ∗P < 0.05, ∗∗P < 0.01 indicates significant difference compared with control; #P < 0.05 indicates significant difference compared with LPS treatment group. LPS, lipopolysaccharide; NC, normal control; NHEK, normal human epidermal keratinocyte; siRNA, small interfering RNA.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions

7. Figure 6
Overexpression of Sirt1 increased anti-inflammatory effects in NHEKs. After Sirt1 or LacZ adenovirus transfection, NHEKs were treated with LPS. (a) Sirt1 and p53 acetylation proteins were determined by Western blot. (b) Sirt1 expression was analyzed by immunocytochemistry. Scale bar = 25 μm. Total RNA was extracted to quantify the mRNA expression of (c) TNF-α and (d) IL-6. (e) PGE2 secreted to media was determined by ELISA. (f) Nuclear/cytosolic proteins were detected by Western blot using anti-NF-κB. (g) Scratch assays were photographed under light microscope. Original magnification ×200. (h) MMP-9 activities were examined by gelatin zymography. Bar graph was generated using mean ± standard error of the mean (n = 3). ∗P < 0.05, ∗∗P < 0.01 indicates significant difference compared with control; #P < 0.05 indicates significant difference compared with LPS treatment. Ad-Sirt1, Sirt1-overexpressing adenovirus; LPS, lipopolysaccharide; MMP, matrix metalloproteinase; NHEK, normal human epidermal keratinocyte; PGE2, prostaglandin E2; TNF, tumor necrosis factor.
Journal of Investigative Dermatology  , DOI: ( /j.jid )
Copyright © 2017 The Authors Terms and Conditions