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Real-time QPCR measurements were performed with a Light Cycler https://en.wikipedia.org/wiki/Tiospirone (Roche), using designed primers (GAPDH/HA01844155/156; CD44/H6060F10/F11, HAS2/HA02031251, HAS3/HA01749381/382, TG1/D2066B02/B03, FLG/HA01749379/380), (Sigma-Aldrich, Saint-Quentin Fallavier, France), HAS1/10336-022 (Invitrogen, Cergy-Pontoise, France). After incubating for 5?days in the presence of LR2412, Episkin? samples (four samples per concentration) were embedded in Tissue-Tek OCT compound (Miles, Naperville, IL, USA), and cryosections (7?��m) were prepared (CM3050 cryostat; Leica Microsystems, Rueil-Malmaison, France). Episkin? samples were stained with haematoxylin�Ceosin�Csafran (HES), and epidermal thickness was measured using image analysis with Axiovision software (Zeiss, Sartrouville, France). Immunolabelling was performed using specific antibodies against human Ki67 (M7240, 1/100; Dako, Trappes, France), CD44v3 (3G5, 1/200; R&D system, McKinley, MN, USA), transglutaminase 1 (BT621 1/50; Biomed Technology, Stoughton, MA, USA), filaggrin (SC-70921 1/500; Santa Cruz Biotechnology, Heidelberg, Germany) and a secondary Alexa 488-conjugated antibody (Invitrogen). The nuclei were stained with propidium iodide. Fluorescence microscopy analysis was performed on an Axiovert 135 Zeiss microscope. http://www.selleckchem.com/products/Docetaxel(Taxotere).html The percentages of positive Ki67 cells were determined from more than 10?000 keratinocytes counted per condition. HA was detected after 7?days of treatment via biotinylated hyaluronic acid-binding protein (400763-1A, 1/250; Seikagaku, Abingdon, UK). The bound biotinylated hyaluronan-binding protein was revealed by streptavidin horseradish peroxydase (P0397, 1/500; Dako) reaction for 15?min followed by incubation with diaminobenzidine (Dako) for 7?min. The sections were counterstained using haematoxylin (Dako) according to manufacturer��s protocol. The real-time RT-PCR results show that, while HAS1 expression was not detected, HAS2 and HAS3 were upregulated (P? http://www.selleckchem.com/products/17-AAG(Geldanamycin).html modify CD44 receptor, transglutaminase 1 (TG1) and filaggrin (FLG) expression. These results were confirmed by immunohistology, CD44v3 being the most representative in the skin (Fig.?1b). Stimulation of HAS3 transcription was confirmed by Western blotting (see Data S1) and correlated with an increased HA deposition in the basal and suprabasal layers of Episkin? epidermis (Fig.?1b). Considering the role of HA fragments in stimulating epidermal proliferation and reversing skin atrophy (9,14,15), increased HAS synthesis and HA production upon LR2412 treatment should result in increased epidermal thickness. Indeed, Episkin? epidermal thickness shifts from 69.5?��m?��?6.6 in control to 84.0?��m?��?9.1 (P?