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Fig. 5 | Stem Cell Research & Therapy

Fig. 5

From: DZNep promotes mouse bone defect healing via enhancing both osteogenesis and osteoclastogenesis

Fig. 5

DZNep activated the NF-kB signaling pathway through EZH2-H3K27me3-Foxc1 axis. A BMMs cells were treated with or without 25 nM DZNep for 0, 1, 3, 5 days accompanied the stimulation of RANKL, and the levels of EZH2 and H3K27me3 were determined. B The gray levels of EZH2 and H3K27me3 were quantified and normalized to β-actin using ImageJ. C Expression of the Foxc1 in BMMs treated with M-CSF (30 ng/mL), RANKL (50 ng/mL), and indicated DZNep concentrations for 5 days. D BMMs cells were treated with negative control or different shRna of EZH2, and the levels of EZH2 were determined. E Expression of the Foxc1 in BMMs after treated with negative control or different shRna of EZH2. F BMMs were treated with negative control or shRna-2 of EZH2 and stimulated by M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 5 days. Then the cells were fixed with 4% paraformaldehyde and stained for TRAP. Scale bar = 20 μm. G Quantification of number of osteoclasts, area of osteoclasts. H BMMs were seeded onto the hydroxyapatite-coated Osteo Assay plates and cultured with M-CSF (30 ng/mL), RANKL (50 ng/mL), and with negative control or shRna-2 of EZH2 for 7 days. Scale bar = 20 μm. I Quantification of bone resorption area was performed by ImageJ. J BMMs cells were treated with negative control or different siRna of Foxc1, and the levels of Foxc1 were determined. K BMMs were treated with siRna-NC, siRna-NC with DZNep, or siRna-3 of Foxc1 with DZNep and stimulated by M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 5 days. Then the cells were fixed with 4% paraformaldehyde and stained for TRAP. Scale bar = 5 μm. L Quantification of number of osteoclasts, area of osteoclasts. M BMMs were seeded onto the hydroxyapatite-coated Osteo Assay plates and cultured with M-CSF (30 ng/mL), RANKL (50 ng/mL), and with siRna-NC, siRna-NC with DZNep or siRna-3 of Foxc1 with DZNep for 7 days. Scale bar = 10 μm. N Quantification of bone resorption area was performed by ImageJ. O Expression of the osteoclast-specific genes Nfatc1, c-Fos, Trap, and Ctsk in BMMs treated with siRna-NC, siRna-NC with DZNep, or siRna-3 of Foxc1 with DZNep and stimulated by M-CSF (30 ng/ml) and RANKL (50 ng/ml) for 5 days (*p < 0.05; **p < 0.01; ***p < 0.001; ****p < 0.0001). Data are expressed as median and interquartile range, n = 3–4

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