Molecular phenotypes of <i>in vitro</i> cultured keratinocytes diverge from <i>in situ</i> keratinocytes.

<p>Expression of markers of keratinocyte differentiation in human skin, HaCaT cell line, human Normal Epidermal Keratinocytes (hNEK) and Primary culture of mouse Keratinocytes (mPK). A. Images acquired with a LSM780 confocal microscope (Parameters 2, see <i>Material</i> and <i>Methods</i>) represent 0.9 µm thick slides of immunolabbeled human frozen skin sections. Panels show detection of 1) keratin 5 (green), K5, and PCNA (red), top left panel, 2) keratin 14 (green), K14, and PCNA (red), bottom left panel, 3) keratin 10 (green), K10, and Involucrin (red), INV, top right panel, 4) loricrin (green), LR, and vimentin (red), VIM, bottom right panel. B. Represents the immunodetection of K5 (green), K10 (green) or INV (red) in non-confluent HaCaT cells and hNEK cultured in media completed to 1.8 mM Ca²⁺. Images were acquired with Parameters 2. hNEK populations were not confluent though colonies were formed locally. C. The same as B for isolated and confluent mPK. K5, K10, K14 and LR are labeled in green. D. Panel shows the sensitivity of detection of K10 (green) with 2 different sets of parameters (parameters 1 and 2) of the confocal microscope. With parameters 1, the most sensitive, K10 shows a strong expression in human skin sections (top left panel), a low expression in the most part of hNEK (middle left panel) and a strong expression in a low population of hNEK (bottom left panel). With parameters 2, the less sensitive, K10 is only seen in human skin sections and in a low population of hNEK. E. Images of immunocytofluorescence show the detection of K10 and INV in hNEK cultured with increasing Ca²⁺ concentrations (top panels) or with 2% FCS at different Ca²⁺ concentrations (bottom panels). The confocal microscope was set with Parameters 1. All experiments were reproduced three times independently. Scale bars = 10 µm.