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. 2019 Aug 28;294(42):15395–15407. doi: 10.1074/jbc.RA119.010139

Figure 4.

Figure 4.

Stat3 deficiency impaired osteoclast differentiation. A, BMMs from 4-week-old WT and Stat3Ctsk mice were cultured with M-CSF and RANKL until mature multinucleated osteoclasts formed (5–7 days). TRAP staining was performed to evaluate osteoclasts. B, relative TRAP activity in the culture supernatants of WT and Stat3Ctsk BMMs on day 7 after addition of M-CSF. Five wells in each group were tested in this experiment, and more than three independent experiments were performed. Error bars represent mean ± S.D.; *, p < 0.05. C, levels of STAT3 and CTSK proteins in WT and Stat3Ctsk BMMs. Cells were cultured with M-CSF and RANKL until mature multinuclear osteoclasts formed (5–7 days), and then expression levels were evaluated by Western blotting. D–G, expression of Stat3 and the osteoclast differentiation markers Oscar, Dc Stamp, and Ctsk by WT and Stat3Ctsk BMMs cultured with M-CSF and RANKL until formation of mature multinuclear osteoclasts (5–7 days). Three independent cell isolations were collected, and tests were performed three times. Experiments were repeated more than three times independently. Error bars represent mean ± S.D.; *, p < 0.05. H, BMMs from 4-week-old Stat3fl/fl mice were infected with adenovirus expressing GFP and CRE recombinase and then cultured with M-CSF and RANKL until mature multinuclear osteoclasts formed (5–7 days). TRAP staining was performed to evaluate osteoclast numbers. I, relative TRAP activity in the culture supernatant of BMMs grown in 96-well plates and evaluated on day 7 after addition of M-CSF. Five wells in each group were tested. Error bars represented mean ± S.D.; *, p < 0.05. J, levels of STAT3 and CTSK protein in BMMs infected with Ad-GFP or Ad-CRE, then cultured with M-CSF and RANKL until formation of mature multinuclear osteoclasts was observed (5–7 days), and tested by Western blotting. K–N, Stat3 and osteoclast-specific gene expression of BMMs infected with Ad-GFP or Ad-CRE and then cultured with M-CSF and RANKL until mature multinuclear osteoclasts formed (5–7 days). Three independent cell isolations were collected and were tested three times. Error bars represent mean ± S.D.; *, p < 0.05.