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All-trans Retinoic Acid Induces Differentiation and Apoptosis of Murine Melanocyte Precursors with Induction of the Microphthalmia-Associated Transcription.

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Presentation on theme: "All-trans Retinoic Acid Induces Differentiation and Apoptosis of Murine Melanocyte Precursors with Induction of the Microphthalmia-Associated Transcription."— Presentation transcript:

1 All-trans Retinoic Acid Induces Differentiation and Apoptosis of Murine Melanocyte Precursors with Induction of the Microphthalmia-Associated Transcription Factor  Hidenori Watabe, Yoshinao Soma, Masaru Ito, Yoko Kawa, Masako Mizoguchi  Journal of Investigative Dermatology  Volume 118, Issue 1, Pages (January 2002) DOI: /j x x Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

2 Figure 1 Immunostaining and DOPA reaction of NCC-melb4 cells. Cells were cultured on cover glasses in six-well plates and were immunohistochemically stained as described in Materials and Methods. The cells were positive for TYRP1 (a), TYRP2 (b), and KIT (c), but were negative for EDNRB (d), tyrosinase (e), and DOPA reaction (f). Scale bar: 10 µm. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

3 Figure 2 RT-PCR analysis for RAR mRNA expression in NCC-melb4 cells. Total RNA was reverse transcribed followed by 35 PCR cycles of cDNA using the primers described in Materials and Methods. Lane 1, β-actin; lane 2, RARα; lane 3, RARβ; lane 4, RARγ. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

4 Figure 3 Effects of ATRA on tyrosinase expression and DOPA reaction. NCC-melb4 cells treated with ATRA for 72 h were immunostained for tyrosinase (a) and were tested for DOPA reaction (b). Scale bar: 10 µm. (c) Total RNA extracted from the cells treated with or without ATRA for 72 h was reverse transcribed followed by 34 PCR cycles of cDNA using the primers described in Materials and Methods. Lanes 1, 2, β-actin; lanes 3, 4, tyrosinase. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

5 Figure 4 Morphologic changes after ATRA treatment. Phase-contrast photomicrographs of NCC-melb4 cells were made before (a) and after (b) 72 h of treatment with ATRA. Scale bar: 40 µm. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

6 Figure 5 Electron microscopy and electron microscopic DOPA staining of NCC-melb4 cells. (a) Electron microscopy before ATRA treatment. Arrows indicate stage I melanosomes. (b) Electron microscopic DOPA staining before ATRA treatment showing negative DOPA reaction. (c) Electron microscopy after 72 h of ATRA treatment showing stage III to IV melanosomes in the cytoplasm (arrows). (d) Electron microscopic DOPA staining after 72 h of ATRA treatment showing positive reactions in the Golgi areas and in melanosomes. Scale bar: 1 µm. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

7 Figure 6 Induction of MITF and PKCα by ATRA treatment. RT-PCR analysis of MITF (a) and PKCα (b) mRNA from NCC-melb4 cells treated with or without ATRA for 72 h. (a) Lanes 1, 2, β-actin; lanes 3, 4, MITF. (b) Lanes 1, 2, β-actin; lanes 3, 4, PKCα; lanes 5, 6, PKCβ. (c) Western blot analysis of PKCα from the cells treated with or without ATRA for 72 h. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

8 Figure 7 Growth inhibition by ATRA. NCC-melb4 cells were plated at 2000 cells per well in 96-well plates and were incubated with (a) SCF (□), SCF + 10-6 M ATRA (◊), or SCF + vehicle (○); (b) SCF + various concentrations of ATRA. On days 0, 2, and 5 (a) and on day 5 (b), cells were incubated with alamar BlueTM and the fluorescence was read on a Fluoroskan II microplate reader. Values are means ± SD of six determinations. *p < 0.05; **p < 0.01. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

9 Figure 8 Electron microscopy findings of apoptotic cells after ATRA treatment. Apoptotic cells with scattered fragments of condensed nuclei (closed arrow) contained melanosomes at advanced stages (open arrows). Scale bar: 1 µm. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

10 Figure 9 Increased apoptosis after ATRA treatment. NCC-melb4 cells before (a) and after (b) 72 h of ATRA treatment were stained with the Apop Tag in situ apoptosis detection kit. Scale bar: 20 µm. (c) DNA fragmentation assay. Agarose gel electrophoresis was used to detect internucleosomal DNA cleavage in NCC-melb4 cells with or without ATRA for 72 h. Lane 1, control; lane 2, ATRA-treated cells; lane 3, ladder marker. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

11 Figure 10 Flow cytometric analysis of ATRA-treated cells. Contour diagram of FITC-Annexin V/PI flow cytometry of the cells incubated with ATRA (d, e, f) or vehicle alone (a, b, c) for 1 d (a, d), 3 d (b, e), and 5 d (c, f). The lower left quadrants of the panels show the viable cells, which exclude PI and are negative for FITC-Annexin V binding. The upper right quadrants contain the nonviable, late apoptotic cells or necrotic cells, positive for FITC-Annexin V binding and for PI uptake. The lower right quadrants represent the early apoptotic cells, which are FITC-Annexin V positive and PI negative, demonstrating cytoplasmic membrane integrity. This diagram is representative of three separate experiments with similar results. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

12 Figure 11 Induction of caspase-3 by ATRA treatment. NCC-melb4 cells treated with (b) or without (a) ATRA for 72 h were immunostained for caspase-3 as described in Materials and Methods. Scale bar: 20 µm. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

13 Figure 12 Decreased expression of bcl-2 after ATRA treatment. NCC-melb4 cells treated with or without ATRA for 72 h were analyzed by Western blotting for their expression of bcl-2. Journal of Investigative Dermatology  , 35-42DOI: ( /j x x) Copyright © 2002 The Society for Investigative Dermatology, Inc Terms and Conditions

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