C2c12 cell line

C2C12 cell line (ATCC, Manassas, VA, USA) was cultured in low glucose Dulbecco’s modified Eagle’s medium (DMEM) (Invitrogen, Carlsbad, CA, USA) supplemented with 10% fetal bovine serum (FBS; Invitrogen) and penicillin–streptomycin (Invitrogen). Cells were treated with vehicle (0.1% DMSO) or 3 μm SRT2104 for 24 h and then harvested for protein and Western blotting using methods detailed elsewhere.

All procedures were approved by McGill’s University’s Animal Care Committee (protocol # 2012– 7127 approved 01 July 2018) and complied with the ethical guidelines of the Canadian Council on Animal Care. Bone marrow- cells and compact-bone-derived osteoblasts (CB-Ob cells) were isolated from 4–6 weeks old C57BL/6 (Charles River) as previously described [20 (link)]. CB-Obs were plated at 10⁴ cells/cm² in osteoblast differentiation medium (αMEM supplemented with 10% FBS, 1% sodium pyruvate, 1% penicillin-streptomycin and 50 µg/mL ascorbic acid [AA]) and cultured for 2–3 days prior to experiments. The C2C12 cell line (ATCC CRL-1772) stably transfected with BMP-2 (C2-Ob cells) was plated at 10⁴ cells/cm² in DMEM (supplemented with 10% FBS, 1% sodium pyruvate, 1% penicillin-streptomycin) and cultured for 2–3 days before experiments. Bone marrow cells were plated at a density of 5 × 10⁴ cells per cm² in osteoblastic differentiation medium supplemented with 2 mM β-glycerol phosphate (βGP) to promote mineralization and media was refreshed every 2–3 days. The osteoblast phenotype was assessed at days 14 and 28. The absence of mycoplasma contamination was verified in cryo-preserved stocks of C2-OB cells using a PCR-based detection kit.

C2C12 cell line was purchased from ATCC (Rockville, MD, USA) and cultured in Dulbecco’s modified Eagle’s medium (DMEM: Gibco, Carlsbad, CA) supplemented with 10% fetal bovine serum (FBS: Hyclone Laboratories, Logan, UT) at 37 °C and 5% CO₂. After cells reached confluence, they were induced to myogenic differentiation by replacing 10% FBS with 2% horse serum (Gibco, Carlsbad, CA). For cell proliferation detection, cells were transfected with Sirt1 AS lncRNA, miR-34a, Sirt1 AS lncRNA plus miR-34a and pCDNA3.1 empty plasmids using Lipofectamine 2000 (Life Technologies, Shanghai, China) according to the manufacturer’s instructions when the cells reached 50–60% confluence after seeding for 12 hours. Twenty-four hours post-transfection, the cells were harvested and preserved at −80 °C for further proliferation assay. For cell differentiation analysis, cells were seeded in six-well plates with culture medium and transfected with above plasmids on the first day of myogenic differentiation. Forty-eight hours post-transfection, they were harvested and preserved at −80 °C for further myogenic differentiation detection.