Figure 3

TG2 knockdown increases Blimp1 expression through the activation of the NF-κB signaling pathway. BMMs were transfected with control or TG2 siRNA and cultured for the indicated number of days in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). (a) The mRNA levels of TG2 and Blimp1 were assessed by real-time PCR. *P < 0.05; **P < 0.005 versus control siRNA. (b) The protein levels of TG2, Blimp1, c-Fos, and NFATc1 were analyzed by Western blotting. C, control siRNA; T, TG2 siRNA. (c) Cytoplasmic and nuclear fractions were obtained from cells cultured for two days with M-CSF and RANKL. Western blotting was performed to detect Blimp1 and NFATc1. PARP (full length) and α-tubulin were used as loading controls for cytoplasmic and nuclear extracts, respectively. CTL, control. (d) Cells cultured for two days after siRNA transfection were deprived of serum and factors for 5 h. Cells were then re-stimulated with RANKL (500 ng/ml) for the indicated times. Whole cell lysates were subjected to Western blot analysis with anti-phospho-IκB, -IκB and anti-phospho-p65 antibodies. (e) Nuclear fractions were prepared from cells stimulated as in (d). A p65 transcription activity assay was performed as described in Materials and methods. *P < 0.05 between indicated groups. (f) Seven predicted NF-κB binding sites on the promoter of Blimp1 are shown (upper). Chromatin immunoprecipitation assay with control IgG or p65 antibody was performed with RAW264.7 cells transfected with control or TG2 siRNA. PCR was performed using primer sets to detect the seven predicted sites (lower). C, control siRNA; T, TG2 siRNA. (g) BMMs transfected with control or TG2 siRNA were cultured with or without BAY 11-7085 (5 μM) for two days in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). Cells were then serum-starved for 5 h and re-stimulated with RANKL (500 ng/ml) for 5 min. Cell lysates were prepared and subjected to Western blotting. C, control siRNA; T, TG2 siRNA. (h) siRNA-transfected BMMs were cultured with or without BAY 11-7085 (5 μM) for two days in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). Western blotting was performed to assess the levels of the indicated proteins. C, control siRNA; T, TG2 siRNA. (i) siRNA-transfected BMMs were cultured in the presence of M-CSF (30 ng/ml) and RANKL (100 ng/ml). The mRNA levels of Irf8 and MafB were assessed by real-time PCR. **P < 0.005 versus control siRNA. C, control siRNA; T, TG2 siRNA. Full length Western blots are presented in Supplementary Figure 9.