Fig 2: Metformin-mediated autophagy downregulation via BECN1 and BNIP3 is E2F1 dependent. A Cell lysates of osteoclast precursors were used to examine the expression of E2F1, BNIP3 and LC3 proteins by western bolt. After overexpression of E2F1 (Lv-E2F1), osteoclast precursors were treated with metformin (0, 20 µM) for 5 d in the presence of 30 ng/mL M-CSF and 50 ng/mL RANKL. B The quantitative analysis of immunoblots relative to GAPDH protein level in (A). C After overexpression of E2F1 (Lv-E2F1), osteoclast precursors were treated with metformin (0, 20 µM) for 24 h, and immunofluorescence staining for LC3 was performed. D Quantification of the LC3 puncta per cell in (C). E After overexpression of E2F1 (Lv-E2F1), osteoclast precursors were treated with metformin (0, 20 µM) for 24 h, immunofluorescent staining (IF) analyses of osteoclast precursors using anti-BECN1 antibody. (F, G) After osteoclast precursors were treated as in (A), TRAP staining was performed and TRAP-positive multinucleated osteoclasts (= 3 nuclei) were counted. Values displayed are means ± SD of 3 independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001, ns, non-significant)

Fig 3: E2F1-siRNA administration rescues OVX-related bone loss in vivo. Mice were treated with Vehicle (Saline) or E2F1-RNAi (250 nmol/kg) through tail vein twice per week for 4 weeks after four weeks following ovariectomy or sham surgery. A Representative immunohistochemical staining for E2F1 in distal femur sections from sham (n = 3), OVX + vehicle (n = 3) and OVX + E2F1 RANi (E2F1 siRNA; n = 3). B Representative immunohistochemical staining for BNIP3 in distal femur sections from sham (n = 3), OVX + Vehicle (n = 3) and OVX + E2F1 RANi (n = 3). C Micro CT analysis of the distal femur from sham, OVX + Vehicle and OVX + E2F1 RNAi group. D Calculations of bone mineral density (BMD), bone value/total value (BV/TV), bone surface area/total value (BS/TV) and trabecular number (Tb.N). E Histological H&E staining of distal femur sections. (F) Quantitative histomorphometric assessment of trabecular number (Tb. N) based on H&E-stained femur sections. G Histological TRAP staining of femur sections. H Quantitative assessment of osteoclasts number based on TRAP-stained tibial sections. Values displayed are means ± SD of 3 independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001)

Fig 4: Metformin inhibits osteoclast differentiation via decreasing autophagy. A Immunofluorescence staining for LC3 in osteoclast precursors isolated from the femur and tibia of sham group mice, OVX group mice, and OVX + MET group mice. B Quantification of the LC3 puncta per cell in (A). C, D After infection with tandem GFP-red fluorescent protein (RFP)-LC3 lentivirus, osteoclast precursors were treated with metformin and/or rapamycin (10 nM) for 24 h in presence of 30 ng/mL M-CSF and 50 ng/mL RANKL, and then the fluorescence was observed by fluorescence microscopy and quantitative analysis was performed. E, F metformin and/or rapamycin were used to treat osteoclast precursors for 24 h, and then the formation of autophagosomes and/or autolysosomes in osteoclast precursors were observed under transmission electron microscopy. G After metformin or/and rapamycin, TRAP-stained multinucleated osteoclasts. H Quantitative analysis of the area of TRAP-positive osteoclasts. Data are presented as means ± SD of 3 independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001, ns, non-significant)

Fig 5: Metformin suppresses RANKL-induced osteoclast differentiation in vitro. A schematic diagram of the experiment. B Chemical structural formula of metformin. C Osteoclast precursors were incubated with different concentrations of metformin for 5 d in the presence of 30 ng/ml M-CSF and 50 ng/mL RANKL. Subsequently, cell viability was evaluated with Cell Counting Kit 8 (CCK8) reagent. D Representative images of TRAP-positive cells after treatment with metformin. E Quantitative analysis of the area of TRAP-positive osteoclasts. F, G Effect of metformin on RANKL-induced osteoclast precursors differentiation at different stages. H, I Pit formation assay of osteoclasts and quantification of the pits area. Data are presented as means ± SD of 3 independent experiments (*p < 0.05; **p < 0.01; ***p < 0.001)