Fig. 3

NBIF helps maintain hBMSCs self-renewal ability. a Heatmap displaying relative expression levels of skeletal progenitor-related genes in hBMSCs cultured in different conditions. The transcriptome data were used. b LEPR relative expression levels (normalized to GAPDH) of hBMSCs, evaluated by quantitative RT-PCR. Data [and also in (d) and (h)] were expressed as mean ± standard error of the mean (SEM) of the fold change across three replicates for each group. P-values were obtained from an unpaired t-test; **P ≤ 0.01, ***P ≤ 0.001. c, d Representative images (c) and quantification data (d) of alkaline phosphatase-positive hBMSC CFUs. CFU formation from hBMSCs was analyzed at passage 1 after 14 days of culture, followed by the alkaline phosphatase activity assay. e Gene set enrichment analysis (GSEA) using the transcriptome data indicates that the IL6_JAK_STAT3 signaling pathway is enriched in NBIF-supplied hBMSCs with FDR < 0.25. f, g Western blot detection of phospho-STAT3(Tyr705) level in BMSCs cultured in different conditions for 7 days. Three replicate were shown for each condition, relative phospho-STAT3(Tyr705) level were shown by pSTAT3/STAT3 ratio and fold change (g). h, i Representative images of osteogenic induction assays (h) and their quantification results (i) of hBMSCs. The hBMSCs were first cultured in medium supplemented with FBS or NBIF, with either negative control siRNA (siNC) or STAT3 siRNA (siSTAT3), for 7 days, followed by osteogenic induction and Alizarin Red staining. Scale bar, 100 μm