Fig. 5

Depletion of MSC membrane cholesterol reduces Akt phosphorylation and osteogenic supplement-induced alkaline phosphatase (ALP) activity. MSCs were treated with standard growth medium (GM), treated with growth medium with osteogenic supplements (OM), or pretreated for 1 hour with methyl-β-cyclodextrin (MβCD) before addition of OM supplements. a, b Immunoblotting (western blot, WB) showed that 10 mM MβCD suppressed phosphorylation of Akt in OM (a), with no change in total Akt levels (b). Molecular weight standards are shown on the left of the immunoblot. These results are representative of three replicates with different MSC sources. c Densitometric analysis of the phosphorylated Akt (p-Akt) signal normalized to total Akt is shown for all three replicates. Values represent mean ± SEM (n =3). d Effect of MβCD treatment on OM-induced ALP activity. Significant suppression of ALP activity was seen at 10 mM MβCD. Values represent mean ± SEM (n = 4). e Three representative images from Live/Dead staining of cells exposed to 10 mM MβCD in replicate wells of samples in (d). Bar = 100 μm. Results shown here are representative of at least three different MSC populations studied, and suggest the requirement of membrane cholesterol for PI3K/Akt signaling and induction of osteogenesis in human MSCs