Fig 2: The overexpression of SLAMF8 promotes the mice femoral fracture healing. (A) Micro-CT images and quantitative analyses (BV/TV, Tb.Th, Tb.Sp and BMD) of femoral fracture healing (n = 5). The arrows indicated the fracture area. *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group. (B) Histological analysis, including Hematoxylin and Eosin (H&E), Safranin O/Fast Green, and Masson’s Trichrome staining, of femoral fracture healing. Scale bars = 200 μm. (C) The expressions of COL1A1 (Red) and OCN (Green) protein at femoral fracture area were performed by immunofluorescence. Scale bars = 50 μm

Fig 3: SLAMF8 was a bone formation related gene. (A) Volcano Plots of normalized gene expressions in GSE176086. (B) Volcano Plots of normalized gene expressions in GSE129165. (C) Venn plot of 31 differentially expressed genes at the intersection of GSE129165 and GSE176086. (D) Correlations between SLAMF8 and RUNX2, COL1A1 expression levels in the cancer cell lines of the CCLE database. (E) Correlations between SLAMF8 and RUNX2, COL1A1 expression levels in the bone relative cancer cell lines of the CCLE database. (F) Correlations between SLAMF8 and RUNX2, COL1A1 expression levels in the osseous and chondromatous neoplasms and miscellaneous bone tumors of the TCGA database. (G) Correlations between SLAMF8 and RUNX2, COL1A1 expression levels in the normal tissues of the GTEx database. The correlation co-efficients (R values) and the p-values were those of the Pearson correlation. (H) GSEA of bone development-related genes, bone mineralization-related genes, regulation of bone mineralization-related genes and regulation of osteoblast differentiation-related genes in SLAMF8_High group and SLAMF8_Low group. The normalized enrichment score (NES) > 1.25, P < 0.05, and false discovery rate (FDR) < 0.05. (I) Endogenous expression of SLAMF8 during osteogenesis. (J) Endogenous expression of SLAMF8 during adipogenesis

Fig 4: SLAMF8 overexpression promoted osteogenesis and inhibited adipogenesis in mBMSCs. (A) The expressions of osteogenesis related genes are examined by qPCR during osteogenic induction in SLA-NC and SLA-OE mBMSCs. (B) The protein levels of osteogenesis related genes were determined by WB during osteogenic induction in SLA-NC and SLA-OE mBMSCs. (C-D) The ALP activity and calcium deposits in SLA-NC and SLA-OE mBMSCs were revealed by ALP staining (after 7 days of osteogenic induction) and ARS staining (after 21 days of osteogenic induction), respectively. Scale bars = 100 μm. (E-F) IF staining for COL1A1 and RUNX2 after 7 days osteogenic differentiation of SLA-NC and SLA-OE mBMSCs. Scale bars = 200 μm. (G) The expressions of Pparγ and C/ebpα were measured by qPCR in SLA-NC and SLA-OE mBMSCs during adipogenesis. (H) The protein levels of PPARγ and C/EBPα were measured by WB in SLA-NC and SLA-OE mBMSCs during adipogenesis. (I) Oil Red O Staining detected the lipid drops in SLA-NC and SLA-OE mBMSCs during adipogenesis. Scale bars = 100 μm. All data were represented mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group

Fig 5: SLAMF8 was a positive regulator of Wnt/β-Catenin signaling pathways in mBMSCs during osteogenesis and adipogenesis. (A) The main signaling pathways in SLA-NC and SLA-OE mBMSCs were examined by WB during osteogenic induction. (B) The activation of β-Catenin in SLA-NC, MOCK and SLA-si mBMSCs during osteogenesis were measured by WB. (C) The activation and localization of SLAMF8 (Red) and β-Catenin (Green) in SLA-NC and SLA-OE mBMSCs after 6 h osteogenic induction was analyzed by confocal microscopy. Scale bars = 20 μm. (D) The activation of β-Catenin in SLA-NC and SLA-OE mBMSCs during adipogenesis were measured by WB. (E) The activation of β-Catenin in SLA-NC, MOCK and SLA-si mBMSCs during adipogenesis were measured by WB. All data were represented mean ± SD (n = 3). *p < 0.05, **p < 0.01, and ***p < 0.001 versus the control group