PI3K inhibition results in reduced chondrocyte differentiation. (A) Mesenchymal cells isolated from E11.5 mouse limb buds were cultured for 3 days and then treated with LY294002 or DMSO as vehicle control. They were stained after 6, 9 and 12 days, respectively for different chondrocyte differentiation markers: Alcian blue for glycosaminoglycans, Alkaline phosphatase activity and Alizarin red for the calcium content. The intensity of these markers is reduced in the LY294002 treated cultures. (B) After 9 and 12 days respectively the Alcian blue content of the micromasses was spectrophotometrically measured at 620 nm, after extraction with 6 M Guanidine hydrochloride. We noticed decreased absorbance levels for the LY294002 treated cultures. (C) Measurements of Hoechst fluorescence intensity (excitation/emission: 350/450 nm) showed no significant difference in the DNA content between the LY294002 and DMSO treated micromass cultures. (D, E) RNA was isolated from the micromass cultures after 6 and 9 days of culture and real time analysis was performed. The relative transcript levels for col2a1 and col10a1 were decreased in the LY294002 treated micromasses compared to the vehicle control.
Ulici et al. BMC Developmental Biology 2008 8:40 doi:10.1186/1471-213X-8-40