Fig 2: Overexpression of DLX5 in C-PCs stimulate hypertrophy, ossification, catabolism markers and increases apoptosis. (A) Relative mRNA expression levels of DLX5 gene, chondrogenic markers (B) COL1, (C) COL2 as well as hypertrophy and ossification-related markers (D) BGLAP, (E) COL10, (F) RUNX2, and catabolic proteinase (G) MMP13 in DLX5 overexpressed C-PCs vs. control C-PCs. N ≥ 3; **, p ≤ 0.01; ***, p ≤ 0.005 relative to control C-PCs. (H) Caspase-3/7 immunofluorescent staining of DLX5 overexpressed C-PCs vs. control C-PCs. Scale = 250 µm.

Fig 3: Enhanced bone fracture healing in Stat5a-/- mice.a Representative longitudinal sections of fractured femurs from wild-type and Stat5a-/- mice at 2 and 4 weeks post-fracture. 6-week-old male mice were used for the fracture model. Scale bar, 4 mm (n = 16 per each group). b Quantitative analysis of newly formed callus volume at 2 and 4 weeks post-fracture. c–f Representative histological analysis of paraffin sections of calluses from wild-type and Stat5a-/- mice at 2 and 4 weeks post-fracture stained with (c) Safranin O/Fast Green staining for cartilaginous bone callus. d Quantitative analysis of remaining cartilaginous callus area at 2 and 4 weeks post-fracture (n = 8 per each group). Scale bar, 0.5 mm. e Masson’s Trichrome staining for total callus. f Quantitative analysis of total callus area at 2 and 4 weeks post-fracture (n = 8 per each group). Scale bar, 0.5 mm. g Immunohistochemistry against DLX5 at 2 weeks post-fracture of wild-type and Stat5a-/- mice in the fractured femoral section. Scale bars, 100 μm (n = 5 for each group). All error bars indicate ±SEM. *P <0.01; **P <0.01; ***P <0.001

Fig 4: Protein expression of osteogenesis-associated genes. Runx2, Dlx5, ALP, Ibsp, VEGF, osteonectin and collagen I protein levels in BMSCs, BMSCs + EPCs, BMP2 BMSCs and BMP2 BMSCs + EPCs cultures by western blot analysis. Runx2, runt related transcription factor 2; Dlx5, distal-less homeobox 5; ALP, alkaline phosphatase; Ibsp, integrin binding sialoprotein; VEGF, vascular endothelial growth factor; collagen I; type I collagen BMSCs, bone marrow stromal cells; EPCs, endothelial progenitor cells; BMP2, bone morphogenic protein 2.

Fig 5: Induced osteoblast differentiation in Stat5a-/- mBMSCs via upregulation of DLX5.a ALP staining of 10-week-old wild-type and Stat5a-/- mBMSCs at day 0, as indicated. Scale bar, 60 μm. b Relative ALP activity assay results at days 0 in wild-type and Stat5a-/- mBMSCs. c and d Alizarin Red S staining (c) and quantitative analysis (d) of at day 8 after induction of osteogenesis in 10-week-old, 20-week-old, and 30-week-old wild-type and Stat5a-/- mBMSCs. e Protein levels of DLX5 in 10-week-old, 20-week-old, and 30-week-old male wild-type and Stat5a-/- mBMSCs using western blotting at 5 days after osteogenesis. f mRNA levels of Dlx5 in wild-type and Stat5a-/- mBMSCs at day 3 after the induction of osteogenesis. g mRNA levels of osteoblast-related genes Alp, Bsp, and Opn at day 3 and mRNA level of Ocn at day 7 after the induction of osteogenesis. h–j Relative mRNA (h) and protein levels (I and j) of DLX5 depending on exogenously increased Stat5a expression in wild-type and Stat5a-/- mBMSCs. pcDNA-mStat5a was transfected in Stat5a-/- mBMSCs. mRNA and protein levels of Dlx5 were checked at day 3 and 5 after osteogenesis, respectively. Osteogenesis was induced after overexpression of Stat5a by transfection. k The osteoblast differentiation of Stat5a-/- mBMSCs upon overexpression of STAT5A using Alizarin Red S and Von Kossa staining at day 8 after induction of osteogenesis. l Quantification of Alizarin Red S staining in Stat5a-/- mBMSCs. Each experiment was performed in triplicate (n = 3). All error bars indicate ±SEM. *P < 0.01; **P < 0.01; ***P < 0.001