Calmodulin-Like Protein Upregulates Myosin-10 in Human Keratinocytes and Is Regulated during Epidermal Wound Healing In Vivo Richard D. Bennett, Amy.

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Calmodulin-Like Protein Upregulates Myosin-10 in Human Keratinocytes and Is Regulated during Epidermal Wound Healing In Vivo Richard D. Bennett, Amy S. Mauer, Mark R. Pittelkow, Emanuel E. Strehler Journal of Investigative Dermatology Volume 129, Issue 3, Pages 765-769 (March 2009) DOI: 10.1038/jid.2008.288 Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 1 CLP and Myo10 expression are correlated. (a) Western blot showing Myo10 and CLP expression during keratinocyte differentiation in vitro. Confluent keratinocyte cultures were stimulated to undergo terminal differentiation by the addition of 1.2mM CaCl2 to the medium and withdrawing growth factors. Cells were lysed at the indicated time (days postconfluence) and proteins separated by SDS–PAGE before western blotting for Myo10 (top), CLP (middle), and GAPDH (as loading control). (b) Representative western blot of subconfluent (undifferentiated) keratinocytes either untransfected (-, control) or transiently transfected with a CLP expression vector (TO-CLP, +) and probed for Myo10, CLP, and GAPDH. Journal of Investigative Dermatology 2009 129, 765-769DOI: (10.1038/jid.2008.288) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 2 CLP expression in normal skin and Myo10 localization to filopodial tips in keratinocytes. (a) Immunohistochemical detection of CLP in a paraffin section of normal human skin. The corresponding H&E stained section is shown on the right. Scale bar=200μm. (b) Immunofluorescence staining for Myo10 (left panel) and actin (Texas-Red phalloidin, right panel) in keratinocytes grown on glass coverslips. Arrowheads indicate filopodial tip localization of Myo10. Scale bar=10μm. Journal of Investigative Dermatology 2009 129, 765-769DOI: (10.1038/jid.2008.288) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 3 Myo10 and CLP are involved in keratinocyte migration in vitro. (a) Wound-healing scratch assays to determine keratinocyte migration. Keratinocytes were transiently transfected with negative control siRNA or siRNA directed against CLP or Myo10 as indicated. Scratches were made in confluent monolayers of transfected cells and photographed at time 0 and 4hours later. The contours of the wound edges are outlined to better visualize the open space. (b) Quantitative analysis of at least three independent scratch assays as in (a) showing average wound area covered in 4hours±SD. *Difference significant at P≤0.05. Journal of Investigative Dermatology 2009 129, 765-769DOI: (10.1038/jid.2008.288) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions

Figure 4 CLP is upregulated and shows increased peripheral localization in basal and suprabasal keratinocytes during wound healing. Immunohistochemical staining for CLP was performed on paraffin sections of a healing wound. A composite (tiled) micrograph is shown on the bottom, and one tile (framed) is shown enlarged on top. CLP staining is increased in basal and suprabasal cells immediately adjacent to the wound bed (white arrows, top tile) and concentrated in the cell periphery. Dark arrows in the bottom micrograph indicate normal epidermis exhibiting regular CLP expression. Scale bar=200μm. Journal of Investigative Dermatology 2009 129, 765-769DOI: (10.1038/jid.2008.288) Copyright © 2009 The Society for Investigative Dermatology, Inc Terms and Conditions