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. 2021 Jun 23;8:666295. doi: 10.3389/fmolb.2021.666295

FIGURE 2.

FIGURE 2

Ti CM induced osteoclastogenic differentiation in Raw 264.7 cells. (A) TRAP staining of osteoclasts (100×), osteoclast formation, and morphology were observed by light microscopy. (B) The number of TRAP-positive cells with two or more nuclei was counted after 9 days of Ti CM or Rankl (50 ng/mL) treatment of Raw 264.7 cells. Rankl treatment is taken as a positive control. (C) Quantification of TRAP activity. D(a–d) Ti CM induced the relative mRNA expression of osteoclast markers (TRAP, Rankl, MMP-9, and CSTK) after 9 days of Ti CM or Rankl treatment of Raw 264.7 cells. (E) NFATc1 reporter plasmid was transiently transfected to Raw 264.7 cells. The Ti CM treatment was done for 24 h, and reporter activity was analyzed from the cell lysate. Rankl treatment is taken as a positive control. Data are expressed as mean ± SD. n = 3. *p < 0.05, **p < 0.01, and ***p < 0.001 vs. control.