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Regulation of Guanylate-Binding Protein Expression in Interferon-γ-Treated Human Epidermal Keratinocytes and Squamous Cell Carcinoma Cells  Nicholas A.

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Presentation on theme: "Regulation of Guanylate-Binding Protein Expression in Interferon-γ-Treated Human Epidermal Keratinocytes and Squamous Cell Carcinoma Cells  Nicholas A."— Presentation transcript:

1 Regulation of Guanylate-Binding Protein Expression in Interferon-γ-Treated Human Epidermal Keratinocytes and Squamous Cell Carcinoma Cells  Nicholas A. Saunders  Journal of Investigative Dermatology  Volume 112, Issue 6, Pages (June 1999) DOI: /j x Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Time-dependent action of IFN-γaction in human keratinocytes. (A) Northern blot of changes in GBP mRNA expression at various intervals following treatment of preconfluent cultures of human keratinocytes with IFN-γ (1000 units per ml) for 36 h. For reference the mRNA expression of differentiation marker cornifin (SQ37) and proliferation-associated gene (E2F-1) is provided. Normalization for loading inequalities is provided by comparison with GAD expression. Thirty micrograms of total RNA was used in all lanes. (B) Total cellular protein was extracted from preconfluent cultures of human keratinocytes at various times following IFN-γ treatment (1000 units per ml). Twenty micrograms of protein was electrophoresed and blotted. A western blot of changes in GBP expression is shown. For comparison changes in cdc2 (proliferation) and SQ37 (differentiation) are also shown. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Concentration-dependent induction of GBP protein expression. Subconfluent cultures of normal human epidermal keratinocyte cells were treated with varying concentrations of IFN-γ for 36 h. GBP protein was detected by immunoblotting and densitometry of the exposed film. Data are presented as arbitrary densitometer units. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Protein synthesis independence and transcriptional dependence of GBP mRNA induction. Normal human epidermal keratinocyte cells were pretreated for 1 h with vehicle (–), 2.5 μg per ml cycloheximide (Chx) or 250 ng per ml actinomycin D (Actino D) followed by an 8 h treatment with 300 units IFN-γ per ml (IFN-γ) as appropriate. Total RNA was extracted and 30 μg total RNA electrophoresed and blotted. GBP mRNA expression is shown. GAD mRNA expression is shown for comparison of loading inequalities. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Specificity of IFN-γaction in normal human epidermal keratinocyte cells. Preconfluent cultures of normal human epidermal keratinocyte cells were treated for 36 h with TGF-β1 (100 pM), IFN-α (300 units per ml), phorbol ester (TPA, 80 nM per ml), IFN-γ (300 units per ml) or no treatment (proliferation). Total RNA was extracted and 30 μg total RNA electrophoresed and blotted. The expression of GBP mRNA is shown as is the differentiation marker SQ37. The expression of IFN-γ receptor mRNA is also shown (IF[capnu]γ rec). Normalization for loading is provided by comparison with GAD mRNA expression. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 GBP mRNA expression is suppressed by modulators of differentiation. Preconfluent normal human epidermal keratinocyte cells were treated with 150 units IFN-γ per ml, 100 pM TGF-β1, 1 μM RA or no treatment (Prolif.) either alone or in combination as shown. RNA was extracted following a 36 h treatment and 30 μg total RNA electrophoresed and blotted. GBP and transglutaminase type I (TGase I) expression are shown as a northern blot. The regulation of differentiation by TGF-β1 and RA is demonstrated by transglutaminase type I mRNA expression. Densitometry of TGase I (black bar) and GBP (hatched bar) expression is also shown normalized for GAD expression. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 GBP mRNA expression in squamous cell carcinoma cell lines. Preconfluent cultures of normal human epidermal keratinocyte cells and the various named cell lines were treated in the absence or presence of 300 units IFN-γ per ml. Total RNA was extracted and 25 μg RNA electrophoresed and blotted. mRNA expression of the proliferation-associated gene E2F-1, the differentiation-specific gene SQ37 is also shown. Normalization for loading inequalities is provided by comparison with GAD mRNA expression. Portions of this figure have been presented in a recent review article (Saunders et al. 1996). Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 The effect of protein kinase inhibitors on IFN-γaction in normal human epidermal keratinocytes. Preconfluent normal human epidermal keratinocyte cells were treated with 100 units IFN-γ per ml for 36 h with varying concentrations of (A) H7 or (B) staurosporine (ST). RNA was then isolated, blotted and probed for GBP (▪), SQ37 (•) or transglutaminase type I (○). All data have been normalized against GAD expression. Data are expressed as a percentage of the expression induced by IFN-γ alone. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 1999 The Society for Investigative Dermatology, Inc Terms and Conditions

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