Fig. 1.

Notch signaling represses chondrogenesis in limb bud cell micromass culture. Limb bud cells were cultured in micromass and infected with NICD1 lentivirus before being harvested for Alcian blue staining, western blot and RT-PCR analysis. (A) Overexpression of NICD1 resulted in decreased cartilage nodule formation at days 3, 5, and 7, compared to GFP-lentirvirus infected controls (Co). Scale bars, 100 µm. (B) Western Blot showed a significant increase of NICD protein expression at days 3, 5 and 7 following NICD1 lentiviral infection. (C) Fold change in protein level of NICD1 in Western blots was determined by measuring band intensity. (D) Real time PCR data showed a significant increase of Notch target gene Hes1 expression at days 3, 5 and 7 following NICD1 lentiviral infection compared to GFP-lentirvirus infected controls (Co). (E) In control cells infected with GFP-lentivirus, Col2α1 expression was increased from day 2 to day 5, with maximal increase at day 5. (F) Agc1 expression was increased at days 3, 5 and 7, with maximal increase at day 7. Both Col2α1 and Agc1 expression were significantly down regulated by overexpression of NICD1 at days 3, 5 and 7. Data are means ± s.d. of three independent experiments performed in duplicate and the control gene expression level at day 3 was set at 1. (*, P < 0.05 compared with control at same time point.)