FIGURE 2.

DKK3 regulates ESC-SMC differentiation. For the shutdown of DKK3 experiment, ES cells were infected with lentiviruses containing shCtl or shDkk3 (10⁷ TU/ml complemented with 10 μg/ml polybrene) after 2 days of pre-differentiation. The cells displayed different morphologies (A) 2 days post-infection and were subjected to Q-PCR (B) and Western blot (C) analysis for SMC specific markers. Quantification of Western blot images (mean ± S.E. of three individual experiments) revealed a significant down-regulation of SMC specific markers as well as DKK3 at the protein level (D). For the overexpression of DKK3, ES cells were cultured in DM for 2 days before infection with adenovirus particles containing Ad-Null or Ad-Dkk3. Samples were harvested 2 days after infection. Q-PCR (E and F) revealed a further induction of SMC markers, such as SM22, SMA, SMMHC after overexpression of DKK3 and Western blot (G) showed up-regulation of SMC markers. In addition, ELISA of the supernatant after overexpression of DKK3 revealed a considerable increase in DKK3 cytokine release (H). The final results were normalized to every 10⁶ cells (data shown as the mean ± S.E. of three individual experiments).