Figure 4. TM deficiency enhanced HMGB1 translocation and secretion, bone resorption, and ovariectomy-induced bone loss.

(A) After 24-hour treatment, whole-cell lysates were fractionated into nuclear (N) and cytosolic (C) fractions. The localization of HMGB1 was analyzed by western blotting. Evaluation of HMGB1 secretion by western blot analysis in primary cultured macrophages from (B) TM^flox/flox and LysMcre/TM^flox/flox mice, and from (C) TM^WT/LeD and TM^LeD/LeD mice. GST was added as internal control. CM, conditioned medium. Results of bone resorption assay were obtained by measuring the (D) pit area in fluoresceinamine-labeled chondroitin sulfate/calcium phosphate-coated plates and (E) fluorescence intensity in conditioned medium. (F) Detected HMGB1 levels in serum by ELISA assay after 4 weeks of ovariectomy. (G) Bone loss in tibia was detected by μCT scanning after 4 weeks of ovariectomy. Dashed lines indicated the cross sections. (H) Quantitative results of trabecular bone volume (BV), total bone volume (TV), and BV/TV in (G). n = 5 each group. (I) Immunohistochemical staining for cathepsin K in the tibia (brown). bar = 100 μm. (J) Osteoclast number/bone surface (N.Oc/BS, N/mm) were measured. Statistics were performed by Student’s t-test. *P < 0.05, **P < 0.01.