Decreased growth of the LY294002 treated tibiae. (A) E 15.5 mouse tibiae, cultured for 6 days in the presence of LY294002 or DMSO, were analyzed by immunohistochemistry, at the end of the time course. Phosphorylation of Akt at Ser473 was found to be reduced under PI3K inhibition with LY294002 (10 μM). Black arrows show cells expressing phosphorylated Akt. (B) The difference between the length of the tibiae in the beginning and at the end of the time course represents the bone growth and it was found to be significantly reduced in bones treated with LY294002 (45% reduction) or PI3-K α inhibitor IV (500 nM) (35% reduction) compared to DMSO. (C) At the end of culture period the tibiae were also stained with Alcian blue/Alizarin red. We notice decreased length of both proximal (gp1) and distal (gp2) growth plates of tibiae treated with LY294002 or PI3-K α inhibitor IV. (D) Measurements of the growth plates and mineralized area (min) length confirmed the reduction of these under PI3K inhibition with LY294002. (E) When the gp1, gp2 and min were expressed as ratio of the entire bone length there is an increased ratio of the mineralized area in the LY294002 treated tibiae.