Fig. 5

Pro-osteogenic activity of the soluble (SF) and vesicular (VF) fractions of CM-SMURF1. (A) Top Panel. Expression of osteogenic markers, alkaline phosphatase activity and mineralization at day 16 of osteogenic differentiation after preconditioning with either the soluble (SF) or vesicular (VF) fractions of the hCM-SMURF1. RUNX2 and ALPL expression, as well as alkaline phosphatase activity and mineralization show significant increases in SF of hCM-SMURF1 in relation to hCM-Ctrl. VF do not impact RUNX2, ALPL or alkaline phosphatase activity, however significant enhancement in mineralization is observed. Results are presented as means ± SEM (n = 3). *: p-value < 0.05; **: p-value < 0.01; ***: p-value < 0.001. (B) Alizarin Red staining of representative samples reflecting the stimulation of mineralization in the SF and VF of hCM-SMURF1. (C). Volcano plot showing differently expressed proteins between hCM-Ctrl and hCM-SMURF1 for both SF and VF. Statistical significance was determined using an adjusted t-test against CM-Ctrl, with a p-value threshold of 0.05 and a fold change of 2 or greater. (D) Validation of relative expression by quantitative PCR of 2 genes overexpressed in soluble fraction (SPARC and CCN2) and 2 genes overexpressed in vesicular fraction (PREPL and FMOD) of CM-SMURF1. Results are presented as means ± SEM. *: p-value < 0.05. (n = 5)