Figure 1

TG2 regulates RANKL-induced osteoclast differentiation. (a) BMMs were cultured for two days with M-CSF (30 ng/ml) alone or with M-CSF plus RANKL (100 ng/ml) to obtain pOCs. The mRNA expression levels of TG1-TG7 and FXIIIA were analyzed by RT-PCR. (b) BMMs were transfected with control or TG2 siRNA for 18 h. The efficiency of knockdown was determined by real-time PCR and Western blotting. TG2 mRNA expression was normalized using the HPRT housekeeping gene, and values indicating the fold-change from control are shown. β-actin was used as a protein loading control. CTL, control. (c) BMMs transfected with control or TG2 siRNA were cultured with M-CSF plus RANKL for three days. Cells were stained for TRAP activity. Scale bar, 200 μm. (d) TRAP-positive MNCs (≥3 nuclei) were counted as osteoclasts. The surface area per osteoclast was measured using the Osteomeasure program. (e) The mRNA levels of c-Fos, and NFATc1 were measured by real-time PCR. (f) Whole cell lysates were subjected to Western blot analysis with anti-TG2, -c-Fos, and -NFATc1 antibodies. C, control siRNA; T, TG2 siRNA. (g) Nuclear fractions were obtained from cells cultured for two days and subjected to an NFATc1 transcriptional activity assay. CTL, control. (h) BMMs transfected with control or c-Fos or NFATc1 siRNA were subjected to real-time PCR analyses, and values indicating the fold-change from control are shown (left). BMMs transfected with indicated combination of siRNAs were cultured with M-CSF plus RANKL for four days. Cells were stained for TRAP activity (right upper). Scale bar, 100 μm. TRAP-positive MNCs (≥3 nuclei) were counted as osteoclasts (right lower). **P < 0.005 versus control siRNA or between indicated groups. Full length gels and Western blots are presented in Supplementary Figure 9.