Overexpression of Sox9 inhibits keratinocyte differentiation.

<p>(A) Expression of exogenous Sox9 in keratinocytes cultured in vitro. Cells were transduced with adenovirus expressing GFP-tagged Sox9 (Ad/GFP-Sox9) at 10 multiplicity of infection (MOI) for overnight. Cells were replenished with fresh medium and incubated for 2 d. The expression of exogenous Sox9 was observed under the fluorescent microscopy. Adenovirus expressing GFP (Ad/GFP) was used as a negative control. (B) Effect of Sox9 on the expression of differentiation markers. Keratinocytes were transduced with adenovirus expressing GFP-Sox9 at the 10 MOI for overnight. Cells were replenished with fresh medium and incubated for the indicated time points, in a low calcium concentration (<0.1 mM) or a high calcium concentration (1.2 mM). The protein level was verified by Western blot. Involucrin and loricrin are the markers for keratinocyte differentiation. Sox9 bands represent the exogenously expressed GFP-Sox9 fusion protein of ∼82 kDa. (C) The effect of Sox9 on involucrin and loricrin promoter activity. Keratinocytes were transduced with involucrin-luc or loricrin-luc reporter adenovirus together with GFP-Sox9 expressing adenovirus for overnight. Next day, cells were replenished with low calcium (0.01 mM) or high calcium (1.8 mM) medium and incubated for indicated time points. Cells were lysed and assayed for luciferase activity. Data are presented as fold induction and SEM, measured from three independent experiments.