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Impact of Bcl-2 and Ha-ras on Keratinocytes in Organotypic Culture

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Presentation on theme: "Impact of Bcl-2 and Ha-ras on Keratinocytes in Organotypic Culture"— Presentation transcript:

1 Impact of Bcl-2 and Ha-ras on Keratinocytes in Organotypic Culture
Maryse Delehedde, Song H. Cho, Rebecca Hamm, Shawn Brisbay, Timothy J. McDonnell  Journal of Investigative Dermatology  Volume 116, Issue 3, Pages (March 2001) DOI: /j x Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Immunoblot analysis of Ha-ras and bcl-2 protein in representative HaCaT transfectant clones. Total protein extracts were electrophoresed on 12.5% SDS-PAGE, electroblotted, and hybridized with an anti-Ha-ras antibody specific for the valine 12 mutation and antibcl-2 antibodies as described in Materials and Methods. Actin served as a control for equal loading. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 Light microscopy of HaCaT cells grown in organotypic culture. Light microscopy appearance of hematoxylin and eosin stained sections of organotypic cultures generated from HaCaT control cells (A), HaCaT-bcl-2 transfectants (B), HaCaT-ras transfectants (C), and HaCaT-ras/bcl-2 transfectants (D); scale bar: 50 μm. These sections are representative of rafts made from the control cells and from a total of 11 clones of bcl-2, ras, and ras/bcl-2 transfected cell lines. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Immunohistochemical evaluation of involucrin expression in organotypic cultures. Two week organotypic cultures of HaCaT control cells (A), HaCaT-bcl-2 transfectants (B), HaCaT-ras transfectants (C), and HaCaT-ras/bcl-2 transfectants (D) were stained using an APAAP technique and an anti-involucrin antibody as described in Materials and Methods. Scale bar: 50 μm. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Immunohistochemical evaluation of cytokeratin 1 and cytokeratin 14 expression in organotypic cultures. Two week organotypic cultures of HaCaT control cells (A, E), HaCaT-bcl-2 transfectants (B,F), HaCaT-ras transfectants (C, G), and HaCaT-ras/bcl-2 transfectants (D,H) were stained using horseradish peroxidase diaminobenzidine reaction and anti-K1 (A–D) and cytokeratin 14 (E–H) antibodies as described in Materials and Methods. Scale bar: 50 μm. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Immunoblot analysis of K1, K14, and involucrin protein in HaCaT organotypic cultures. Total protein extracts were electrophoresed on 12.5% SDS-PAGE, electroblotted, and hybridized with antibodies (involucrin, K1, and K14) as described in Materials and Methods. Actin was used as a control for loading. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Immunohistochemical evaluation of PCNA and p21 expression in organotypic cultures. Two week organotypic cultures of HaCaT control cells (A, E), HaCaT-bcl-2 transfectants (B,F), HaCaT-ras transfectants (C, G), and HaCaT-ras/bcl-2 transfectants (D,H) were stained using a horseradish peroxidase technique and anti-PCNA (A–D) and p21 (E–H) antibodies as described in Materials and Methods. Scale bar: 50 μm. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Comparison of proliferative rates and of the expression of the cell cycle proteins in organotypic cultures. (A) The PI was calculated as the percentage of PCNA-positive cells by scoring at least 2000 cells in each of four 2 wk organotypic cultures for each genotype. The data represent the mean ±the standard deviation. Statistical comparisons were calculated using Student's t test. (B) Immunoblot analysis of PCNA, p21cip1/waf1, and cyclin D protein in HaCaT organotypic cultures. Total protein extracts were electrophoresed on 12.5% SDS-PAGE, electroblotted, and hybridized with antibodies for PCNA, p21cip1/waf1, and cyclin D as described in Materials and Methods. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Comparison of apoptotic rates in organotypic cultures before and following ultraviolet irradiation. The AI was calculated as the percentage of TUNEL-positive cells by scoring at least 2000 cells in each of four 2 wk organotypic cultures for each genotype before and 24 h after ultraviolet irradiation (4 kJ per m2). The data represent the mean ±the standard deviation. Statistical comparisons were calculated using Student's t test. Journal of Investigative Dermatology  , DOI: ( /j x) Copyright © 2001 The Society for Investigative Dermatology, Inc Terms and Conditions

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