The small GTPase ARF4 is required for K10 and Notch reporter expression and epidermal differentiation. (A and B) Quantification of K10 or GFP-TNR (Notch reporter) expression in 1°MKs transduced with pooled shRNAs against the indicated ciliary and trafficking proteins. Histograms represent data from two individual experiments where pooled shRNAs were transduced to cultured keratinocytes before differentiation, and the percentage of K10 (+) or GFP (+) cells was quantified after 48-h Ca2+ addition to induce differentiation. (C and D) Images of differentiating 1°MKs transduced with Arf4 or Bbs2 shRNAs and then immunolabeled with antibodies against K10 or tested for Notch reporter activity. DAPI used to visualize nuclei. (E) Example of Arf4-2 shRNA transduced tissue adjacent to nontransduced epidermis. IF is for K10 (green) to mark epidermal differentiation and H2B-RFP (red) to mark transduced cells. (F and G) Example of Arf4-2 or scrambled control shRNA transduced tissue immunolabeled for antibodies against FILAGGRIN or LORICRIN (green). H2B-RFP (red) marks transduced cells. Bars, 30 µm.
