MiR-24 affects proliferation and induces differentiation of primary keratinocytes. (A) Northern blot highlights miR-24 up-regulation in differentiating (diff.) mouse keratinocytes. As a positive control, Northern blot for miR-203 is included. (B) In situ hybridization of skin from newborn mice reveals restriction of miR-24 expression to suprabasal layers. As a reference, hematoxylin–eosin (H/E) staining and anti–β4 integrin immunostaining for the basal lamina are included. epi, epidermis; der, dermis; NG, negative control of in situ hybridization. (C) Overexpression of miR-24 for the indicated time points reduces BrdU incorporation in HEKn. *, P < 0.01; ^#, not statistically significant compared with the control. Histograms of BrdU incorporation show the means ± SD from three independent experiments. (D) Colony formation assay performed on 400 or 2,000 cells plated after 48 h of transfection of scrambled control, miR-24, or anti–miR-24. Images are representative of three independent experiments. Numbers represent the relative percentage of clones (P < 0.01). (E) Morphology of posttransfection keratinocytes grown in proliferating conditions. (F) Morphology of posttransfection keratinocytes grown in differentiating conditions (high confluence). (G) Real-time qPCR analysis of early epidermal differentiation markers K10, K1, and involucrin and the late epidermal differentiation marker filaggrin in cells grown in proliferating conditions. qPCR results are shown as means ± SD from three independent experiments. *, P < 0.01. (H) Real-time qPCR analysis of mRNA of the early epidermal differentiation markers K10, K1, and involucrin (Invol.) and late epidermal differentiation marker filaggrin (Filag.) in cells grown in differentiating conditions. qPCR results are shown as means ± SD from three independent experiments. *, P < 0.01; **, P < 0.05. R.Q., relative quantification; Scr, scramble. Bars: (B, top and middle) 100 µm; (B, bottom) 25 µm; (E and F) 500 µm.