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Fig. 7 | Molecular Medicine

Fig. 7

From: Phosphorylation of BCL2 at the Ser70 site mediates RANKL-induced osteoclast precursor autophagy and osteoclastogenesis

Fig. 7

OCP autophagy and osteoclastogenesis inhibited by BCL2 mutation at S70 was reversed by TAT-Beclin1. A OCPs transfected with WT BCL2 or S70A BCL2 were treated with RANKL along with TAT-Beclin1 (10 μM) or the control peptide (TAT-scrambled) for 8 h in α-MEM with 2.5% FBS. LC3, cleaved PARP, and p62 (soluble and insoluble p62) were detected using western blotting assays. LC3 conversion rate is expressed as the ratio of LC3II to LC3I. B The transfected OCPs were treated with RANKL along with TAT-Beclin1 or TAT-scrambled for 24 h in α-MEM with 2.5% FBS. The autolysosomes (red arrows) in OCPs were observed under TEM. Scale bar, 5 μm or 1 μm. C The histograms showing quantitative results of autolysosomes in B (75 cells from 3 independent experiments). D The transfected OCPs were treated with RANKL along with TAT-Beclin1 or TAT-scrambled for 48 h in α-MEM with 2.5% FBS. Cell apoptosis was examined by flow cytometry of Annexin/PI staining. E The percentages of apoptotic cells (ANNEXIN-positive cells) are shown in the histograms according to the results in D. F The transfected OCPs were treated with M-CSF plus RANKL along with TAT-Beclin1 or TAT-scrambled for 4 days in α-MEM with 5% FBS. Representative images of TRAP-positive multinucleated cells in each group. Scale bar, 200 μm. G, H Quantitative results regarding mature osteoclasts (more than 3 nuclei) or large osteoclasts (more than 5 nuclei) in F. The experiments were replicated at least three times. Data are presented as the mean ± SEM from three independent experiments. ***P < 0.001, **P < 0.01

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