Fig. 4

Osteogenic supplement-induced Akt phosphorylation is modestly enhanced in Cav-1 knockdown MSCs. MSCs transfected with control (Ctrl) or Cav-1 (Cav) small interfering RNA (siRNA) were serum starved overnight, and then stimulated with osteogenic supplements (OM). Immunoblot analysis of cellular proteins harvested at the indicated time points after stimulation with OM showed that Akt is phosphorylated after 5 minutes incubation in OM (top panel). There is a moderate increase in Akt phosphorylation in MSCs transfected with Cav-1 siRNA and incubated in OM for 5 minutes and 40 minutes compared with MSCs transfected with control nontargeting siRNA. This result is representative of three replicates with different MSC sources. The densitometric data for all replicates are presented in the bar graph (bottom panel), which shows the mean p-Akt/Akt signal for each sample relative to control samples in the same experiment at t = 0. Densitometric quantification of bands was achieved using NIH Image J software. Values represent mean ± SEM (n =4). The increase in p-Akt signal in MSCs transfected with Cav-1 siRNA and incubated in OM was statistically significant at 40 minutes