Gadd45a Activation Protects Melanoma Cells from Ultraviolet B-Induced Apoptosis Caroline Fayolle, Julie Pourchet, Claude Caron de Fromentel, Alain Puisieux,

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Gadd45a Activation Protects Melanoma Cells from Ultraviolet B-Induced Apoptosis Caroline Fayolle, Julie Pourchet, Claude Caron de Fromentel, Alain Puisieux, Jean-François Doré, Thibault Voeltzel Journal of Investigative Dermatology Volume 128, Issue 1, Pages 196-202 (January 2008) DOI: 10.1038/sj.jid.5700963 Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 Gadd45a inactivation prevents proliferation of melanoma cells. M4Be melanoma cells were either mock transfected or transfected with the pSuper-Retro vector (Brummelkamp et al., 2002) containing a small hairpin sequence allowing downregulation of Gadd45a (pSR-sh45-2 and pSR-sh45-6) or a non-targeting sequence (pSR-shSc). After 2 weeks of puromycin selection, cells were stained with Giemsa. Journal of Investigative Dermatology 2008 128, 196-202DOI: (10.1038/sj.jid.5700963) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 Gadd45a downregulation leads to increased UVB sensitivity. (a) Detection of Gadd45a by Western blot analysis of control and UVB (100J/m2)-treated M4Be cells and derived clones containing either a non-targeting shRNA (pSR-shSc) or Gadd45a-specific shRNAs (pSR-sh45-2 and pSR-sh45-6). Loading of proteins was standardized by β-actin revelation. (b) Flow cytometry analysis of M4Be cells and derived clones following 100J/m2 UVB irradiation. Asynchronous cultures of M4Be cells were exposed to 100J/m2 UVB and harvested 24hours after irradiation. Experiments were performed three times with similar results. A corresponds to the percentage of cells in subG1 and was calculated using CELLQUEST software (Becton Dickinson, France). Journal of Investigative Dermatology 2008 128, 196-202DOI: (10.1038/sj.jid.5700963) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Gadd45a downregulation increases Caspase-3 activation by UVB. (a) Western blot analysis of Pro-caspase-3 and active Caspase-3 intracellular levels following exposition of M4Be cells and derived clones to 100J/m2 UVB. Loading of proteins was standardized by Ku-80 revelation. (b) Caspase-3 activity was evaluated using a Caspase-3 Fluorescent Assay Kit from Clontech (Becton Dickinson, Palo Alto, CA). Presented results correspond to the ratio between detected fluorescence and the total protein concentration determined by the Bradford assay. Journal of Investigative Dermatology 2008 128, 196-202DOI: (10.1038/sj.jid.5700963) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 Gadd45a prevents the activation of the mitochondrial apoptotic pathway. Western blot analysis of Bcl-xL intracellular level following exposition of M4Be cells and derived clones to 100J/m2. Loading of proteins was standardized by Ku-80 revelation. Journal of Investigative Dermatology 2008 128, 196-202DOI: (10.1038/sj.jid.5700963) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 5 Gadd45a inactivation sensitizes melanoma cells to therapeutic agents. (a) M4Be cells and derived clones exposed to DTIC (400μg/ml) or Cisplatin (8μg/ml) were stained with Giemsa 24hours after treatment. (b and c) Cell survival was evaluated by Trypan blue exclusion 24hours after exposure of M4Be cells and derived clones to (b) DTIC or (c) Cisplatin. The percentage of surviving cells was obtained by enumerating Trypan blue-negative cells versus the total number of cells. Journal of Investigative Dermatology 2008 128, 196-202DOI: (10.1038/sj.jid.5700963) Copyright © 2008 The Society for Investigative Dermatology, Inc Terms and Conditions