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. 2012 Oct 15;199(2):347–363. doi: 10.1083/jcb.201203134

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© 2012 Amelio et al.

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Figure 8. — Overexpression of PAK4, Tks5, and ArhGAP19 cDNAs abolishes the effects of miR-24. (A) Silencing of miR-24 targets reduces in vitro keratinocyte migration. 48 h after transfection, confluent HEKn were subjected to the in vitro scratch wound assay. Cell migration was observed into the scratch at the time indicated. Micrographs are taken at 0 and 24 h after scratch. Red bars denote the distance between the edges of the scratch wounds. Bar, 500 µm. (B) Histogram shows the relative percentage of distance (Dist.) covered by migration (migr.) 24 h after scratch. *, P < 0.001. (C) Transwell migration assay confirmed the reduced migration ability of siRNA-transfected keratinocytes. *, P < 0.001. (D) Specific silencing of PAK4, Tsk5, or ArhGAP19 reduces human primary keratinocyte BrdU incorporation. Histogram shows the relative percent reductions of BrdU-incorporating cells. (E) Specific silencing of PAK4, Tks5, or ArhGAP19 increases keratinocyte differentiation as evaluated by real-time qPCR of specific markers (K10, K1, and involucrin). (F) cDNA of miR-24 targets were introduced into miR-24–transfected keratinocytes by retrovirus infection. Expression of miR-24 targets abrogates miR-24 induction of differentiation as evaluated by real-time qPCR. (G) Expression of miR-24 targets abrogates miR-24–mediated AJ formation as evaluated by confocal analysis of E-cadherin staining. Bar, 50 µm. (H) Histogram shows quantification of AJs of the experiment shown in G. *, P < 0.01. (I) Proposed model for the role of miR-24 in keratinocyte differentiation showing that miR-24 acts on pro-proliferation and actin cable dynamic factors. miR-24 inhibits proliferation repressing Myc, E2F2, etc. expression and induces differentiation by inhibiting the actin cytoskeletal modulators PAK4, Tks5, and ArhGAP19. The overall effect of miR-24 on actin dynamics results in reduced cell motility and directional migration, in changes in cell shape, and in increased formation of AJs, promoting keratinocyte differentiation. Error bars show means ± SD. EV, empty vector; Scr, scramble.