Figure 5. RhoE ablation suppresses growth and commitment to differentiation in vitro and enhances the proliferative phenotype in vivo.

A, SCC4, SCC13 and SCC15 cells were depleted of RhoE expression using siRNA and Keratin 1 mRNA was analyzed 48 hrs later using real time RT-PCR. B, Primary HKC transfected with control or RhoE siRNA were kept under growing conditions (time point 0) or induced to differentiate by suspension or the indicated times. Keratin1 mRNA levels were analyzed by real time RT-PCR. C, Knockdown of RhoE inhibits the Notch1-mediated induction of Keratin 1 and Involucrin. Primary HKC under growing adherent conditions were transfected with control or two different RhoE siRNAs, and 48 hours later infected with adenoviruses AdGFP or AdN1IC. 24 hours later commitment of keratinocytes to differentiation was analyzed by Keratin 1 or Involucrin mRNA levels. D, HKC transfected with control or RhoE siRNA were treated as in C, and analyzed for RhoE, Keratin 1 and Involucrin protein levels by Western blot using tubulin as a loading control. E, Human HKC transfected with control or RhoE siRNA were injected into immunodeficient nude mice at the dermal-epidermal junction. To minimize individual animal variations, the same mice were injected in parallel with control siRNA transfected keratinocytes. 7 days later cysts were harvested, and tissues were processed. F, The same cysts from above experiments were stained with a Keratin 5 specific antibody (green staining in the right panel). Shown are multilayer cysts with well-differentiated keratinocytes building a cornified envelope in the control samples and a large “basal” layer of proliferating keratinocytes with increased Keratin 5 expression (green staining) and decreased Keratin 10 expression (red staining) in the RhoE siRNA tissues. Nuclei were stained in blue (Hoechst staining). Error bars represent Standard Deviations, calculated based on three independent measurements.