Fig. 7.
Integrin-αv heterodimers are required for skin tissue generation but not tissue maintenance. (A) Experimental setup. Human keratinocytes that had been infected with TRIPZ-shintegrin-αv and sorted by using flow cytometry were induced with doxycycline (dox) at various time points in organotypic culture (indicated with arrows) – 4 days prior to seeding, 2 days prior to seeding, day of seeding, 2 days post seeding. (B) Morphological analysis of organotypic tissue in the doxycycline-inducible timecourse experiment described in A. Representative images were taken from two independent experiments, each performed in triplicate. (C) Western blot from tissue lysates that were not treated with dox, or treated with dox for 12 days. OTC, organotypic culture. (D) Quantification of tissue thickness of samples shown in B, measured in mm (mean±s.d.). Representative analysis from two independent experiments. Each experiment comprised three independent organotypic tissues. P=0.00015 using one-way ANOVA. (E) Quantification of BrdU+ basal epidermal cells from the samples shown in B (mean±s.d.). Representative analysis from two independent experiments. Each experiment comprised three independent organotypic tissues. P=7.52×10−8 using one-way ANOVA. (F) Morphological analysis of organotypic tissue treated with inhibitors (suffix ‘i’) of the indicated proteins at the indicated time points. (G) Quantification of tissue thickness measured in mm and PCNA+ basal cells from samples shown in F (mean±s.d.). n=3 independent organotypic tissues. P=0.00879 (upper left), P=0.423 (upper right), P=0.0157 (lower left), P=0.407 (lower right) using one-way ANOVA. (H) Western blot from tissue lysates generated from keratinocytes that had been infected with the indicated shRNAs. *P<0.05; **P<0.005; ***P<0.0005; NS, not significant. Scale bars: 200 μm.