Fig 2: SCRG1/BST1 preserves osteogenic differentiation of hMSCs after ex vivo expansion.(a and b) UE7T-13 cells were cultured in osteogenic differentiation medium (ODM) containing 500 ng/mL (a), or various concentrations of rhSCRG1 (b, 5–500 ng/mL). After 2 weeks, the cells were evaluated for extra cellular matrix mineralization by alizarin red staining (a). Alizarin red was extracted with 10% cetylpyridinium chloride and absorbance was measured at 540 nm (b). (c) UE7T-13 cells were cultured in ODM with 500 ng/mL rhSCRG1 and 1 μM PI3K inhibitor LY294002 (PI3K), MEK inhibitor U0126 (MEK), JNK inhibitor SP600125 (JNK), and FAK inhibitor I (FAK) for 1 week. Total RNAs were extracted and qRT-PCR was performed with tissue non-specific (liver/bone/kidney) alkaline phosphatase (ALPL)-specific oligonucleotide primers. mRNA expression was normalized to GAPDH and results are expressed as fold-increase or decrease relative to the control. (d) Primary cultured hMSCs (passage number #5) were subcultured ten times in the presence (passage number #15, +rhSCRG1) or absence (passage number #15, −rhSCRG1) of 500 ng/mL rhSCRG1. The cells were evaluated for extra cellular matrix mineralization as in (b). In b–d, data are presented as mean ± SD. * p < 0.05 was considered significant.

Fig 3: SCRG1 receptor BST1 relays ERK and PI3K signals in hMSCs.(a) UE7T13 cells were transfected with siRNA for BST1 (siBST1) or HAS2 (siHAS2). mRNA expression of BST1 (left panel) and HAS2 (right panel) was measured by qRT-PCR and normalized to GAPDH; results are expressed as fold increase or decrease relative to the control (Ctrl). Data are presented as mean ± SD. *p < 0.05 was considered significant. (b) UE7T-13 cells were transfected with siBST1 or siHAS2. After transfection for 48 h, the cells were serum-starved overnight and then stimulated with 500 ng/mL rhSCRG1. The cells were washed twice with ice-cold PBS and lysed in RIPA buffer. Samples were separated by SDS-PAGE and analyzed by western blotting. Although cropped blots were used, the gels were run under the same experimental conditions. (c) UE7T-13 cells were transfected with pAdSCRG1-FLAG. After 1 week, the cells were treated with 0.5 mM dithiobis-sulfosuccinimidylpropionate and then lysed in ice-cold RIPA buffer. Lysates were incubated with anti-FLAG M2 Agarose Affinity Gel and proteins were eluted by adding Laemmli sample buffer (IP: FLAG). Eluted proteins were analyzed by western blotting. Total cell lysate (Cell Lys) and 5-fold concentrated conditioned medium (CM) were used as loading controls. Although cropped blots were used, the gels were run under the same experimental conditions.

Fig 4: SCRG1/BST1 preserves self-renewal potential and the expression of stem cell markers in hMSCs even after long-term culture.(a–c) Primary cultured hMSCs (passage number #5) were subcultured ten times in the presence (passage number #15, +rhSCRG1) or absence (passage number #15, −rhSCRG1) of 500 ng/mL rhSCRG1. These primary hMSCs under long-term culture conditions were used for the experiments in a–c. (a) The cells were cultured in growth medium on 96-well culture plates for 5 days. After incubation, proliferation was evaluated by WST-1 assay. (b) Expression of cell surface CD271/LNGFR (left column), CD90/THY-1 (middle column), and CD106/VCAM1 (right column) was analyzed by flow cytometry with FITC- or PE-conjugated specific antibodies. Specific antibody (red) and isotype control IgG (blue) are shown. (c) mRNA expression of POU5F1 (left panel) and NANOG (right panel) were analyzed by qRT-PCR and normalized to GAPDH; results are expressed as fold-increase or decrease relative to the control. In a–d, data are presented as mean ± SD. *p < 0.05 was considered significant.