Figure 7

TG2-deficient BMMs show enhanced osteoclast differentiation in vitro. (a,b) BMMs obtained from WT and TG2 KO mice were cultured with M-CSF (30 ng/ml) and RANKL (100 ng/ml) for three days. Cells were then stained for TRAP. Scale bar, 100 μm. TRAP-positive MNCs were counted, and the surface area per osteoclast was measured. *P < 0.05; **P < 0.005 versus WT cells. (c,d) BMMs were cultured with M-CSF and RANKL for the indicated number of days. The mRNA levels of Blimp1 were determined by real-time PCR. *P < 0.05; **P < 0.005 versus WT cells. Cell lysates were analyzed by Western blotting with the indicated antibodies. (e) BMMs from WT and TG2 KO mice were treated with M-CSF and RANKL for two days. Cells were stained for NFATc1 and laminB. Anti-laminB staining marked the nuclear envelope. Cells with nuclear NFATc1 staining were counted. Scale bar, 20 μm. *P < 0.05 versus WT cells. (f) BMMs were cultured with M-CSF and RANKL for one day. The mRNA levels of Irf8 and MafB were determined by real-time PCR. *P < 0.05; **P < 0.005 versus WT cells. Full length Western blots are presented in Supplementary Figure 10.