Fig. 6

Cholesterol oxidase (CHOD) treatment enhances MSC osteogenesis. a MSCs were treated for 1 hour with or without CHOD, and then caveolae were isolated by sucrose gradient flotation. Fractions expected to contain caveolae (see Fig. 3) were immunoblotted for caveolin-1 (Cav-1). Results show that CHOD pretreatment displaces Cav-1 from buoyant caveolae membrane rafts. b, c MSCs were treated with standard growth medium (GM), treated with growth medium with osteogenic supplements (OM), or pretreated for 1 hour with CHOD before addition of growth medium with osteogenic supplements (CHOD). b After continuous treatment for 10 days with medium change every 3 days, Alizarin red staining revealed that OM-induced matrix mineralization was accelerated in CHOD-treated MSCs (quadruplicate cultures shown). c Immunoblotting showed that 5 minutes after initial CHOD treatment, phosphorylated Akt (p-Akt) levels increased, while total Akt (Pan Akt) remained unchanged. These results are representative of three replicates with different MSC sources. d Densitometric analysis is shown for all three replicates. Values represent mean ± SEM (n =3). This confirmed a significant increase in p-Akt/Akt levels upon CHOD treatment, suggesting that pretreatment with CHOD enhances OM-mediated induction of PI3K/Akt signaling in MSCs. Ctrl control