Bnip3

mRFP-LC3 (21075), tfLC3 (21074), and LAMP1-mGFP(34831) were obtained from Addgene (Cambridge, MA, USA). EGFP-LC3, BNIP3, and SNAP29 plasmids were constructed by Gene Chem Co. Ltd (Shanghai, China). Cells were transfected with plasmids using Lipofectamine 3000 Transfection Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s protocol. After 24 h incubation, the transfection mixture was removed and replaced with fresh complete medium. The target sequence of BNIP3 shRNA (5′-ACTGCACTTCAGCAATAAT-3′) was purchased Gene Chem Co. Ltd. To establish BNIP3 knockdown stable cell lines, 293FT cells were co-transfected with lentiviral packaging vectors pLP1, pLP2, and pLP/VSVG (Invitrogen, K4975) along with shBNIP3 or shCon plasmid using Lipofectamine 3000 (Invitrogen, L3000015) according to the manufacturer’s protocols. Forty-eight hours later, supernatant containing the lentivirus was harvested and infected with MCF-7 cells. Cells were subsequently selected with 4 μg/mL puromycin (Sigma, P9620) to establish stable cell lines.

Osteoclast precursors were seeded into six well plates at 4 × 10⁵ cells/well. The cells were transfected with lentiviral particles for overexpression of BNIP3 or E2F1 (Genechem, Shanghai, China) using polybrene in the presence of 30 ng/mL M-CSF. After 48 h and washing three times to stop the infection, cells were incubated with vehicle or metformin in the presence of 30 ng/mL M‐CSF and 50 ng/mL RANKL.