Three-dimensional (3D) reconstruction images were generated, and analyses were performed using the Metamorph software (Molecular Devices, Tokyo, Japan, RRID:SCR_002368). Ex vivo inhibitor treatment Whole mice paw skin samples were treated with the following inhibitors for 1 hr at 4C: EDTA, pan-PKC inhibitor Go6983 (Tocris Bio, NS13001 Bristol, UK) and myristoylated pseudosubstrate Inhibitor (myr PSI; Calbiochem, Billerica, Massachusetts, USA) before staining. neonatal and aged skin in distinct ways. Our study indicates that COL17 could be an important target of anti-aging strategies in the skin. DOI: http://dx.doi.org/10.7554/eLife.26635.001 and control IFE skin samples from or littermates (Control) at P1 (n?=?5) and P20 (n?=?4). Scale bar: 20 m. Quantitation of the number of epidermal NS13001 layers and epidermal cell counts. The values are shown as relative ratios NS13001 to the controls. (b) PH3 staining at P1 and P20. Scale bar: 20 m. The number of epidermal basal cells positively labeled for PH3 per mm epidermis (n?=?4). BM, basement membrane. (c) PCNA and BrdU labeling at P1. Scale bar: 20 m. Quantitation of PCNA- (n?=?5) and BrdU-positive basal cells (n?=?4). The values are shown as relative ratios to the controls. (d) Quantitative RT-PCR (qRT-PCR) of and mRNAs (n?=?5). (e) Loricrin and cleaved caspase-3 staining (representative images from 3 mice). Scale bar: 20 m. BM, basement membrane. (f) An in silico model of the epidermal cell proliferation upon the reduced adhesion of committed progenitor cells to the BMZ. The details are described in the Material and Methods. The data in all of the histograms are NS13001 the means SE. *0.01 p 0.05, **0.001 p 0.01, ****p 0.0001. Students t-tests. DOI: http://dx.doi.org/10.7554/eLife.26635.003 Figure 1figure supplement 1. Open in a separate window Barrier function assay of and (n?=?4). (b) Dye permeabilization with Toluidine blue (representative images from three and control mice (n?=?4). The data are the meansSE. *0.01 p 0.05, **0.001 p 0.01, Students t-tests. DOI: http://dx.doi.org/10.7554/eLife.26635.004 Figure 1figure supplement 2. Open in a separate window Proliferative ability of the back skin IFE from mice and NHEKs treated with siRNAs.(a) PH3- and PCNA-positive cells in the knockdown efficiency in NHEKs. The left panel shows COL17 immunoblotting of lysates from NHEKs treated with siRNAs. The right panel shows the qRT-PCR results of (n?=?3). (cCd) Cell proliferation curve (c) and slope (d) of NHEKs treated with siRNAs (n?=?3). (eCf) Colony formation assay of NHEKs treated with siRNAs. Gross appearance (e), total colony number (f-left) and the percentage of colonies that were larger than 0.5 mm (f-right) (n?=?3). The data are presented as the meansSE. *0.01 p 0.05, **0.001 p 0.01, Students t-tests. DOI: http://dx.doi.org/10.7554/eLife.26635.005 We investigated whether the expression levels of markers of basal cells and differentiated cells were altered in the hyperproliferative IFE of was not altered (Figure 1d, Figure 1figure supplement 1a). The mRNA expression levels of and were somewhat higher in IFE presented hypoplastic hemidesmosomes in accordance with previous observations on the back skin of mice (Nishie et al., 2007) (Figure 1figure supplement 1d). There were no significant differences in the number of inflammatory infiltrates, including CD3+, F4/80+ and Ly-6G+ cells, in the dermis of and mice and control mice (Figure 1figure supplement 2a). The discordance between the paw epidermis and back skin IFE might be explained either by the influence of hair follicle development on the back skin IFE or by the distinct regulation of the IFE at each body site (Rompolas et al., 2016; Roy et al., 2016; Sada et al., 2016). We also investigated cell-intrinsic properties due to NS13001 COL17 defects using cultured normal human epidermal keratinocytes (NHEKs). The cell proliferation rates of NHEKs treated with siRNAs were slightly decreased (Figure 1figure supplement 2bCd), which is compatible with reduced proliferation of cultured keratinocytes derived from mice (Tanimura et al., 2011), and the colony-forming abilities Rabbit Polyclonal to PNPLA6 of these cells were similar to those of control cells (Figure 1figure supplement 2eCf). These data indicate that the proliferation potential of and (((mice. The LacZ-positive area that was indicative of active Wnt signaling in the IFE was significantly diminished in the ins-Topgal+:mice (Figure 2e, Figure.