1. The Suppressor of Cytokine Signaling-3 Is Upregulated in Impaired Skin Repair: Implications for Keratinocyte Proliferation 
Itamar Goren, Andreas Linke, Elke Müller, Josef Pfeilschifter, Stefan Frank 
Journal of Investigative Dermatology 
Volume 126, Issue 2, Pages (February 2006)
DOI: /sj.jid
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

2. Figure 1
Expression of SOCS-3 in normal skin repair. (a) RNase protection assay demonstrating SOCS-3 mRNA expression in non-wounded back skin (ctrl skin) and wound tissue isolated from Balb/C mice. The time after injury is indicated for each lane. For the RNase protection assay (upper panel), every single experimental time point represents 16 wounds (n=16) isolated from four individual mice (n=4). A GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification of SOCS-3 mRNA (x-fold of control skin) is shown in the lower panel. **P<0.01; *P<0.05. All data points depict means±s.d. obtained from wounds (n=48) isolated from animals (n=12) from three independent animal experiments (n=3). (b) Immunoblot demonstrating the expression of SOCS-3 protein upon skin injury (upper panel). Every experimental time point represents wounds (n=8) isolated from four individual animals (n=4). A Ponceau S staining is given to demonstrate equal loading. Cytokine-stimulated murine primary keratinocytes (mKC-Cyt) and human embryonal kidney (HEK) cells transfected with a human SOCS-3 cDNA (HEKhSOCS-3) served to control the specificity of the antiserum. Immunohistochemical localization of SOCS-3 protein at the wound site on day 5 after injury (lower panel). e, epidermis; g, granulation tissue.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

3. Figure 2
Induction of SOCS-3 expression in human HaCaT keratinocytes. Serum-starved HaCaT keratinocytes were stimulated for the indicated time periods with (a) EGF (10ng/ml) or TGF-α (10ng/ml) (b) DETA-NO (500μM), and (c) a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ). Total cellular RNA was analyzed by RNase protection assay for the presence of SOCS-3 mRNA (upper panels). The stimulation time is indicated for each lane. GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification of SOCS-3 mRNA (x-fold of control) is shown in the middle panels. **P<0.01; *P<0.05. Data represent means±standard deviation (SD) obtained from four independent cell culture experiments (n=4). [35S]methionine-labeled SOCS-3 protein was immunoprecipitated from total cellular lysates after stimulation of cells for the indicated time points and analyzed by SDS gel electrophoresis (lower panels).
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

4. Figure 3
Induction of SOCS-3 in human and murine primary keratinocytes. (a) Quiescent human primary keratinocytes were stimulated with EGF (10ng/ml) or a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ) for the indicated time periods. Total cellular RNA was analyzed by RNase protection assay for the presence of SOCS-3 mRNA. The time after stimulation is indicated for each lane. GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. (b) Quiescent murine primary keratinocytes were stimulated for the indicated time periods with TGF-α (10ng/ml) (c) with a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ) in the presence or absence of the proteasomal inhibitor lactacystin (lac, 1μM). SOCS-3 protein was detected by immunoblot. Detection of actin was used to control for equal loading. One representative experiment out of three is shown.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

5. Figure 4
Overexpression of SOCS-3 inhibits proliferation and promotes differentiation in cultured keratinocytes. (a) Immunoblot demonstrating different SOCS-3 expression levels in SOCS-3-overexpressing HaCaT cell lines OS-8 and OS-16. A mock-transfected HaCaT cell line and human embryonal kidney (HEK) cells transfected with a human SOCS-3 cDNA (HEK/SOCS-3) served as controls. (b) Serum-stimulated proliferation (percent of mock control) of SOCS-3-overexpressing cell lines (OS-8, OS-16) as assessed after 24hours of treatment. **P<0.01; *P<0.05. Data represent means±SD obtained from four independent cell culture experiments (n=4). (c) Total cellular RNA from different control (mock) and SOCS-3-overexpressing cell lines (OS) and Ca2+ (1.8mM)-differentiated HaCaT keratinocytes was analyzed by RNase protection assay for the presence of keratin 1 and keratin 10 mRNA (upper panels). GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. tRNA hybridization was used to control specificity of the antisense probe. A quantification of keratin 1 and 10 mRNA is shown in the lower panels. *P<0.05. Data represent means±standard deviation (SD) obtained from three independent SOCS-3-overexpressing cell lines (n=3). (d) Immunoblot showing expression of loricrin in unstimulated HaCaT (HaCaT), in mock cell lines (mock), in SOCS-3-overexpressing cell lines (OS) after passage 3 or 8 (p3, p8), or in Ca2+ (1.8mm)-differentiated HaCaT (high Ca2+) cells. Detection of actin and staining with Ponceau S were used to control for equal loading. (e) Immunoblot showing phosphorylated STAT-3 (Y-705) after a 20-minute stimulation of a control cell line (mock) and SOCS-3-overexpressing cell lines (OS) with a combination of cytokines (2nM TNF-α, 2nM IL-1β, 100U/ml IFN-γ) or IL-6 (10ng/ml) as indicated. Detection of actin was used to control for equal loading.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

6. Figure 5
Localization of SOCS-3-expressing and proliferating wound margin keratinocytes during normal repair. Directly neighbored serial frozen sections from 3-, 5-, and 7-day wounds as indicated were immunohistologically analyzed for SOCS-3 (left panels) or the Ki67 marker of proliferation (right panels). e, epidermis; g, granulation tissue.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

7. Figure 6
SOCS-3 and Ki67 expression in NO-deficient wound healing. (a) RNase protection assay demonstrating SOCS-3 mRNA expression in non-wounded back skin (ctrl skin) and wounds isolated from C57 wild-type (iNOS+/+) or C57 iNOS knockout (iNOS−/−) mice. The time after injury is indicated for each lane. Every single experimental time point represents 16 wounds (n=16) isolated from four individual mice. GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification (x-fold of control skin) of SOCS-3 mRNA expression in non-wounded back skin (ctrl) and wound tissue isolated from wild-type (C57 iNOS+/+) and iNOS-deficient (C57 iNOS−/−) mice for the indicated time points is shown in the lower panel. **P<0.01; *P<0.05 as indicated by the brackets. All data points depict means±SD obtained from wounds (n=48) isolated from individual animals (n=12). (b) Directly neighbored serial frozen sections from 5-day wounds of iNOS-deficient mice (iNOS−/−) were immunohistologically analyzed for the Ki67 marker of proliferation (left panel) and SOCS-3 (right panel). Total keratinocyte numbers (lower left panel) and percentage of SOCS-3-expressing keratinocytes (lower right panel) in 5-day neo-epithelia of wild-type (iNOS+/+) and iNOS knockout (iNOS−/−) mice are shown. **P<0.01; *P<0.05. Data represent means±SD obtained from wounds (n=4) isolated from individual animals.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions

8. Figure 7
SOCS-3 and Ki67 expression in diabetes-impaired wound healing. (a) RNase protection assay demonstrating SOCS-3 mRNA expression in day-13 wound tissue isolated from PBS- or leptin-treated ob/ob mice. Every single experimental time point represents four wounds (n=4) isolated from individual PBS- or leptin-treated animals (PBS no. 1, 2, 3 or Lep no. 1, 2, 3). GAPDH hybridization is shown as a loading control. As a size marker, 1,000c.p.m. of the hybridization probe was used. A quantification of SOCS-3 mRNA expression in 13-day wounds of leptin- or PBS-treated ob/ob mice is shown in the right panel. **P<0.01. Data depict means±SD obtained from wounds (n=16) isolated from four individual animals (n=4). (b) Frozen serial sections from 13-day wounds of leptin- (left panels) or PBS- (right panels) treated ob/ob mice were analyzed for SOCS-3 protein and Ki67 expression as indicated. e, epidermis; g granulation tissue.
Journal of Investigative Dermatology  , DOI: ( /sj.jid )
Copyright © 2006 The Society for Investigative Dermatology, Inc Terms and Conditions