Fig. 3

BCL2 mutation at S70, not S87, inhibited RANKL-induced osteoclastogenesis. A The working model diagram of site-directed mutagenesis: mutation from S70 to S70A (T-G MUT) or mutation from S87 to S87A (AG-GC MUT). B BCL2 expression in OCPs stably transfected with blank vector, wild-type (WT) BCL2, and two mutant BCL2 constructs (S70A or S87A) was detected using western blotting assays. C After OCPs transfected with WT BCL2 and the two mutant BCL2 constructs were treated with or without RANKL for 20 min in α-MEM with 1% FBS, p-BCL2 (S70), and p-BCL2 (S87) were detected using western blotting assays. D The transfected OCPs were treated with M-CSF plus RANKL for 4 days in α-MEM with 5% FBS. Representative images of TRAP-positive multinucleated cells in each group. Scale bar, 200 μm. E The osteoclastic bone resorption activity caused by OCPs inoculated on bone discs and treated with M-CSF plus RANKL for 6 days in α-MEM with 5% FBS was evaluated by scanning electron microscopy. Scale bar, 400 μm. F Quantitative results showing the number of TRAP-positive multinucleated cells in D. G Quantitative results showing the mean resorption pit area in E. The resorption pit area was normalized to that of WT OCPs. H After the transfected OCPs were treated as described in D, the protein expression of CTSK, MMP9, and TRAP was detected using western blotting assays. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001. ns not significant, Cont the control group without RANKL intervention