Fig. 2

rhRSPO1 in vitro bioactivity. Alkaline phosphatase (ALP) colorimetric assay using a cell lysate obtained from C2C12 cells induced to osteogenic differentiation in the presence of the rhRSPO1 protein. Biological activity of purified rhRSPO1 - 100 ng/mL of rhRSPO1 from the original conditioned media or from various purification steps, and/or 100 ng/mL of WNT3A were used for induction of osteogenic differentiation. OCM: Cl. L1 original conditioned medium; Hep: rhRSPO1 from Step2 of the heparin column purification after buffer exchange; GF: rhRSPO1 from the second purification step using gel filtration (fractions A10 + A11 and A12). DMEM medium containing 5% FBS (C-) and medium conditioned by 293 T cells expressing human recombinant BMP7 were used as negative and positive controls, respectively. The commercially available rhRSPO1 protein (R&D Systems - Cod 4645-RS/CF) was used as reference sample for comparison (Commercial rhRSPO1). The (+) symbol indicates the presence of the listed recombinant protein, whereas traces (−) indicate its absence. ANOVA statistical test (Tukey’s post hoc test) was used and statistical differences were considered to be significant when the p < 0.05