PI3K/Akt signaling is required for MSC osteogenesis. MSCs were cultured in control growth medium (GM) or osteogenic medium (OM) to induce osteogenesis. a Effect of LY294002. MSCs in OM were incubated in the presence or absence of different concentrations of the PI3K inhibitor LY294002, or DMSO vehicle alone. Osteogenesis was assessed by Alizarin red staining for matrix mineralization on day 21 (top panel, triplicate cultures shown), and measuring alkaline phosphatase (ALP) activity on day 4 (bottom panel). Values represent mean ± standard error of the mean (SEM) (n = 4). LY294002 inhibited osteogenesis in a dose-dependent manner, suggesting that PI3K/Akt signaling is required for human MSC osteogenesis. b Effect of siRNA-mediated Akt knockdown. MSCs were transfected with scramble control siRNA (Ctrl) or Akt siRNA before incubation in GM or OM for 4 days and measurement of ALP activity. Akt siRNA treatment significantly reduced ALP activity induced by OM. Values represent mean ± S.E.M (n =4).