Figure 6.

miR-184 Activates the Notch Pathway to Induce KC Differentiation

(A and B) Paraffin sections of wild-type mice at the indicated postnatal day were stained for NICD. Scale bars, 50 μm.

(C) KCs were transfected with pre-miR-184 mimic (PM184), miR184-antagonist (AM184), or the appropriate control (Ctl-PM and Ctl-AM, respectively) and harvested after 48 hr for western blot analysis of the indicated genes.

(D) KCs were co-transfected with a Notch activity reporter plasmid (Hes1-dGFP) and PM184 mimic or miR-184 mutant mimic (C57U) or antagonist (AM184). Forty-eight hours later, cells were trypsinized, and the frequency of GFP-positive cells was quantified by flow cytometry. Data represent the fold increase in GFP-positive cells compared with control transfectants.

(E and F) KCs were co-transfected with PM184 or control (Ctl-PM) and, on the next day, treated with the γ-secretase inhibitor that is required for Notch activation (DAPT) or vehicle (DMSO). After 48 hr, cells were subjected to western blot (E) or quantitative real-time PCR (F) analyses of the indicated genes and proteins.

Data shown are means ± SD from three independent experiments. ^∗p < 0.05 statistically significant by Student's t test. Quantification of western blot analysis from at least three independent experiments (p < 0.05) is shown at the bottom of each panel. The dashed line indicates the dermal-epidermal junction in (A) and the hair follicle in (B). de, dermis; ep, epidermis.