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.