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Human Keratinocytes Respond to Osmotic Stress by p38 Map Kinase Regulated Induction of HSP70 and HSP27  M. Garmyn, A. Pupe  Journal of Investigative Dermatology 

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Presentation on theme: "Human Keratinocytes Respond to Osmotic Stress by p38 Map Kinase Regulated Induction of HSP70 and HSP27  M. Garmyn, A. Pupe  Journal of Investigative Dermatology "— Presentation transcript:

1 Human Keratinocytes Respond to Osmotic Stress by p38 Map Kinase Regulated Induction of HSP70 and HSP27  M. Garmyn, A. Pupe  Journal of Investigative Dermatology  Volume 117, Issue 5, Pages (November 2001) DOI: /j x x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Upregulation of HSP70 and HSP27 mRNA in keratinocytes after osmotic stress and heat shock. (a) Representative northern blot of time-dependent changes in HSP70 and HSP27 mRNA levels in keratinocytes receiving control medium (co) or control medium supplemented with 100, 200, or 300 mM sorbitol. As a positive control, keratinocytes were heat shocked (HS) 20 min on 45°C. Non-human keratinocytes (NHK) were harvested for total RNA at the indicated time-points. Sequential hybridization was performed with cDNA probes for HSP70, HSP27 and GAPDH (normalization for loading consistency). (b) Time course of HSP70 mRNA abundance or (c) HSP27 mRNA abundance in sorbitol treated (concentration in mM) or HS keratinocytes as measured by densitometric analysis of the Northern blot shown in (a). Results are expressed as a percentage of the respective level of mRNA expression for the same transcripts in control treated keratinocytes at each time-point, after correction for even loading (hybridization with GAPDH). Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Induction of Hsp70 protein in keratinocytes after osmotic stress and heat shock. Concentration-dependent increase in Hsp70 protein in sorbitol treated (concentration in mM) or heat shocked (HS) keratinocytes as measured by EIA. Exposure time was 4 h. EIA measures nanograms of Hsp70 protein per 1.0 × 10 (4) cells. Results correspond with mean ± SD (n = 6) of one representative experiment. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 p38 MAPK activation by osmotic stress in keratinocytes. Keratinocytes are treated with control medium (co) or control medium supplemented with 200 or 300 mM sorbitol. Total cell protein was extracted at the indicated times, separated by sodium dodecyl sulfate–polyacrylamide gel electrophoresis, transferred to nitrocellulose membranes and immunoblotted with anti-phospho-p38 antibody. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Effect of p38 inhibitor PD on osmotic stress and heat shock-induced increase HSP70 and HSP27 mRNA levels. (a) Representative northern blot of changes in HSP70 and HSP27 mRNA levels in keratinocytes, treated with or without PD (15 µM) for 30 min prior to osmotic shock (200 or 300 mM sorbitol) or control medium. Keratinocytes were harvested for total RNA 16 h after osmotic shock. Sequential hybridization was performed with cDNA probes for HSP70, HSP27, and GAPDH (normalization for loading consistency). The bar graphs show HSP70 (b) and HSP27 (c) mRNA abundance in sorbitol-treated (concentration in mM) keratinocytes as measured by densitometric analysis of the northern blot shown in (a). Results are expressed as a percentage of the respective level of mRNA expression for the same transcripts in control treated keratinocytes (PD = 0 and sorbitol = 0). Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Effect of p38 inhibitor PD on osmotic stress-induced increase in Hsp70 protein levels. HSP70 protein levels in NHK after osmotic shock in the absence or presence of the p38 MAPK inhibitor PD as measured by EIA. Heat shock was added as a positive control. Keratinocytes were treated with or without PD (10, 15, or 20 µM) for 30 min prior to osmotic shock (100 mM sorbitol) or control medium. Exposure time to osmotic shock or control medium was 4 h. EIA measures nanograms of HSP70 protein per 1.0 × 10 (4) cells. Results correspond with mean ± SD (n = 4) of one representative experiment. Journal of Investigative Dermatology  , DOI: ( /j x x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

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