2 amino 2 methylpropanol

Calvaria cells were isolated from 3‐ to 4‐day‐old C57BL/6 mice as described.⁽³⁵⁾ To obtain bone marrow–derived stromal cells, total bone marrow cells pooled from 3–4 C57BL/6 female mice at 2–4 months of age were cultured with 20% FBS, 1% penicillin–streptomycin–glutamine, and 50 μg/ml of ascorbic acid (Sigma) in 10‐cm culture dishes for 5 days. Half of the medium was replaced every 3 days. Cells were then cultured with 10% FBS, 1% penicillin–streptomycin–glutamine, 50 μg/ml of ascorbic acid, and 10mM β‐glycerophosphate (Sigma0Aldrich) for 21 days with or without HBX. Mineralized matrix was stained with 40mM Alizarin Red solution (Sigma‐Aldrich). BrdU incorporation was measured with a Cell Proliferation ELISA Chemiluminescence Kit (Roche Diagnostics). Alkaline phosphatase activity was determined in cells cultured for 7 days, and cells were lysed in 100mM glycine, 1mM MgCl₂, and 1% Triton X‐100 at pH 10 using a buffer containing 2‐amino‐2‐methylpropanol and p‐nitrophenylphosphate (Sigma‐Aldrich). Alkaline phosphatase activity was normalized to total protein concentration, which was measured using a detergent‐compatible kit (Bio‐Rad). For all assays, cells were plated in triplicate.

C2C12 cells were cultured in DMEM supplemented with 10% FBS, 1% each penicillin, streptomycin, and glutamine, and 1% sodium pyruvate. Expression of SIRT1 was knocked down by transduction with lentiviruses encoding shRNA to Sirt1 (NM_019812) according to the manufacturer’s protocol (Sigma-Aldrich, St. Louis, MO, USA). C2C12 cells transduced with a nontarget shRNA (SHC002V) were used as control. After selection with puromycin (2500 ng mL⁻¹) for 14 days, the SIRT1-silenced cells were seeded at 2 × 10⁴ cm⁻² in medium containing 10% FBS. The following day, the medium was replaced with 5% serum-containing medium. Cells were lysed in 100 mm glycine, 1 mm MgCl_2, and 1% Triton X-100 at pH 10. AP activity in cell lysates was determined using a buffer containing 2-amino-2-methylpropanol and p-nitrophenylphosphate (Sigma-Aldrich). Alkaline phosphatase activity was normalized to protein content, which was determined using a Bio-Rad DC protein assay kit (Hercules, CA, USA).