1. Preclinical Studies of a Specific PPARγ Modulator in the Control of Skin Inflammation 
Arianna Mastrofrancesco, Daniela Kovacs, Massimiliano Sarra, Emanuela Bastonini, Giorgia Cardinali, Nicaela Aspite, Emanuela Camera, Philippe Chavatte, Pierre Desreumaux, Giovanni Monteleone, Mauro Picardo 
Journal of Investigative Dermatology 
Volume 134, Issue 4, Pages (April 2014)
DOI: /jid
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression and activation of peroxisome proliferator–activated receptor γ (PPARγ) in GED L-treated normal human keratinocytes (NHKs). (a) Real-time PCR analysis of the expression of PPARγ in NHKs treated with GED L (0.01–1mM) for 6hours. Troglitazone (Tg) treatment (5μM) was used as a positive control. (b) Luciferase activity analysis of cells transfected with pGL3-(Jwt)3TKLuc reporter construct, treated with GED L (0.01–1mM) and Tg (5μM) for 24hours. (c) Real-time PCR analysis of the expression of PPARγ, PPARα, and PPARβ/δ in NHKs treated with GED L (0.5mM) for 6hours (n=3; *P<0.05; **P<0.01; ***P<0.001). (d) NHKs were co-transfected with PPARα and PPARβ/δ small interfering RNAs (siRNAs) or with nonspecific siRNA (siCtr). PPARα and PPARβ/δ levels were evaluated by real-time PCR (*P<0.01). (e) Real-time PCR analysis of the expression of PPARγ in PPARα/PPARβ/δ-deficient cells in response to GED L (0.5mM; ***P<0.001).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Suppression of proinflammatory mediators in response to GED L. Real-time PCR analysis of the expression of IL-6, IL-8, and tumor necrosis factor-α (TNF-α) in normal human keratinocytes (NHKs) stimulated with (a) TNF-α (10ngml−1) and (b) lipopolysaccharide (LPS) (10μgml−1) in the presence of GED L (0.01–0.5mM) for 6hours (P<0.001 vs. untreated control cells; *P<0.05, **P<0.01, ***P<0.001 vs. stimulated cells). (c) Real-time PCR analysis of the expression of cyclooxygenase-2 (COX-2) in NHKs stimulated with LPS (10μgml−1) in the presence or absence of GED L (0.5mM) for 3hours (n=3; °P<0.01 vs. untreated control cell; *P<0.05 vs. stimulated cells). (d, e) IL-6, IL-8, and TNF-α levels in supernatants of NHKs pretreated with 1μM GW9662 for 1hour before the addition of 10ngml−1 TNF-α, 10μgml−1 LPS, and 0.5mM GED L (n=3; °P<0.001 vs. untreated control cells; *P<0.05, **P<0.01, ***P<0.001 vs. stimulated cells).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Suppression of proinflammatory cytokines in response to GED L on normal human keratinocytes (NHKs) and peripheral blood mononuclear cells (PBMCs). IL-6, IL-8, and tumor necrosis factor-α (TNF-α) protein in NHK supernatants treated with (a) TNF-α (10ngml−1) and (b) lipopolysaccharide (LPS; 10μgml−1) in the presence or absence of GED L (0.01–0.5mM) for 24hours. (c) IL-21, (d) IL-23, (e) IL-12, and (f) TNF-α protein levels measured by ELISA assay in PBMC supernatants from psoriatic and healthy subjects treated with LPS (1μgml−1) in the presence of GED L (0.01–0.5mM) for 24hours. Cytokine levels were measured in duplicate for each condition, normalized for protein content (pgmg−1), and expressed as percentage of untreated cells (% with respect to control; §P<0.05; P<0.001 vs. untreated control cells; °P<0.01, °°P<0.001 vs. stimulated cells).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Suppression of the inflammatory response through peroxisome proliferator–activated receptor γ (PPARγ) signaling induced by GED L. (a) Western blotting analysis of IκBα in normal human keratinocytes (NHKs) after treatment with GED L (0.5mM) for 30minutes until 2hours. A representative experiment is shown. (b) Western blotting analysis of IκBα in NHKs after treatment with tumor necrosis factor-α (TNF-α; 10ngml−1) for 30minutes alone or in the presence of GED L. A representative experiment is shown. (c) Immunofluorescence analysis of NF-κB p65 (red) in NHKs stimulated with TNF-α (10ngml−1) for 30minutes alone or in the presence of GED L. Nuclei are stained with 4',6-diamidino-2-phenylindole (DAPI). Bar=20μm. (d) NHKs were transfected with small interfering RNA (siRNA) specific for PPARγ (siPPARγ) or nonspecific siRNA (siCtr). PPARγ level was evaluated by real-time PCR (*P<0.01). (e, f) Western blotting analysis of IκBα in NHKs transfected with PPARγ siRNA or siCtr and stimulated with TNF-α (10ngml−1) and lipopolysaccharide (LPS; 10μgml−1) alone or in the presence of GED L (0.5mM). (g) Real-time PCR analysis of the expression of TNF-α and IL-8 in NHKs transfected with PPARγ siRNA or siCtr and stimulated with LPS (10μgml−1) for 6hours in the presence or absence of GED L (0.5mM) (°P<0.01 vs. untreated control cells; *P<0.05 vs. stimulated cells). (h) Real-time PCR analysis of the expression of K6 in NHKs transfected with PPARγ siRNA or siCtr and stimulated with TNF-α (10ngml−1) for 6hours in the presence or absence of GED L (0.5mM) (§P<0.01 vs. untreated control cells; *P<0.05 vs. stimulated cells). (i) Real-time PCR analysis of the expression of TNF-α and IL-8 in NHKs co-transfected with PPARα and β/δ -siRNA or siCtr and stimulated with LPS (10μgml−1) for 6hours in the presence or absence of GED L (0.5mM) (n=3; °P<0.01 vs. untreated control cells; *P<0.05, **P<0.01 vs. stimulated cells).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
GED L normalizes the altered keratinocyte proliferation/differentiation induced by tumor necrosis factor-α (TNF-α). (a) Immunofluorescence analysis of Ki67 expression (red) in normal human keratinocytes (NHKs) following TNF-α (10ngml−1) in the presence or absence of GED L (0.5 and 1mM). Nuclei are counterstained with 4',6-diamidino-2-phenylindole (DAPI). Bar=20μm. (b) Percentage of Ki67-positive cells after 72hours of treatment. (c) Western blot analysis of p21 expression on NHKs stimulated with TNF-α (10ngml−1) for 48hours in the presence or absence of GED L (0.5 and 1mM). Densitometric scanning of band intensities was used to quantify change of protein expression. A representative blot is shown. (d) Real-time PCR analysis of the expression of K6 in NHKs stimulated with TNF-α (10ngml−1) for 6 and 24hours in the presence or absence of GED L (0.5mM) (n=3; °P<0.01 vs. untreated control cell; *P<0.05 vs. stimulated cells). (e) mRNA transcript level of filaggrin (FLG) and loricrin (LOR) evaluated by real-time PCR in NHKs stimulated with TNF-α (20ngml−1) in high calcium condition for 24hours in the presence or absence of GED L (0.5 and 1mM) (n=3; *P<0.01 vs. untreated control cells; °P<0.05; §P<0.05; *P<0.01 vs. stimulated cells).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
Reduction of IL-21 epithelial hyperplasia, hyperproliferation/differentiation, and inflammatory markers in mice in response to GED L. Skin biopsies were taken from C57BL6 WT mice (n=9) injected intradermally with 500ng IL-21 or vehicle, and topically treated with placebo or GED L (50mM) and killed at day 6. (a) Representative hematoxylin and eosin (H&E)–stained sections. Bar=20μm. (b) Epidermal thickness measured at day 6 (*P<0.05; **P<0.01). (c) Inflammatory infiltrate measurement. Real-time PCR analysis of the expression of (d) peroxisome proliferator–activated receptor γ (PPARγ), (e) IL-21, (f) tumor necrosis factor-α (TNF-α), and (g) K6. (h) Epidermal proliferation was evaluated by immunohistochemical analysis of proliferating cell nuclear antigen (PCNA). Real-time PCR analysis of the expression of (i) K1, (j) involucrin, and (k) loricrin. Data are shown as mean value±SE (*P<0.05; **P<0.01).
Journal of Investigative Dermatology  , DOI: ( /jid )
Copyright © 2014 The Society for Investigative Dermatology, Inc Terms and Conditions