Ity Animal Care and Use Committee.Gene transfection of murine mesenchymal stem cell line MC3T3EMethodsBriefly 2 ? 105 cells/well were seeded into 6-well plates and incubated overnight, then exposed to mouse Nampt shRNA (Catalog#: CSTVRS TRCN0000101275, Sigma) or pLKO.1 non-mammalian shRNA control lentiviral particles (Catalog#: SHC002H, Sigma) with 8 /mL of polybrene for 24 h. Following the transduction, cells were selected with 800 ng/mL puromycin (Catalog#: ant-pr-1, InvivoGen, San Diego, CA, USA) for 7 days. Puromycin resistant, stably transfected cells were used for further experiments.Alkaline phosphatase (ALP) enzyme staining and quantification, alizarin red stainingStaining of ALP activity was performed with BCIP/NBT substrate solution (Catalog#: B1911, Sigma Aldrich), according to the manufacturer’s instructions. PubMed ID:https://www.ncbi.nlm.nih.gov/pubmed/26509685 Calcium deposition was visualized by alizarin red S (catalog#: A5533, Sigma Aldrich) staining [13]. Cells were cultured in 24-well plates for 2 weeks in OBM, fixed in ice-cold 70 ethanol for 60 min, and incubated with alizarin red (2 , pH 4.2) for 10 min at room temp prior to microscopy. An average of 200 cells/well were counted to calculate the percentage of ALP and alizarin red positive cells.Ling et al. Cell Biosci (2017) 7:Page 3 ofPNPP (p-nitrophenyl phosphate disodium salt, Catalog#: 34045, Pierce, Rockford, IL, USA) was used to quantify the ALP activity in cell cultures [13]. Cells were plated at 20,000/well in 6-well plates and cultured in OBM for 4 days. The cells were lysed in 500 of M-PER mammalian protein extraction reagent without protease inhibitors (Catalog#: P8340, Sigma), followed by incubation (20 lysate) with 100 of PNPP solution in 96-well plate at room temperature for 30 min. Then 50 of 2 N NaOH was added to stop the reaction. The blank control was 20 of M-PER reagent and 100 of PNPP solution. The absorbance was measured at 405 nm in a kinetics ELISA reader (BioTek, Winooski, VT, USA). The results were normalized with the protein concentration of the cell lysates.Isolation of RNA, qPCR, Western blot, and NAD/NADH analysisTotal RNA was isolated from MC3T3-E1 cells with a mirVanaTM miRNA Isolation Kit (Catalog#: AM1561, ThermoFisher Scientific, Waltham, MA, USA) according to the supplier’s instructions. RT-PCR was performed with the cDNA synthesis catalyzed by Superscript III (Catalog#11752-250, ThermoFisher) and PCR amplification using Runx2 specific primers (Forward: 5-CCCAGCCAC CTTTACCTACA-3, Reverse: 5-TATGGAGTGCTGCT GGTCTG-3) synthesized by Integrated DNA Technologies (IDT, Coralville, IA, USA). Western blots were performed as described previously [17]. Briefly, an equal amount (20 ) of protein per each sample was analyzed by SDS polyacrylamide gel electrophoresis and transferred to PVDF membrane. The membrane was incubated with Anti-Nampt antibody (Catalog#: AG-20A-0034, Santa Cruz Bio., Santa Cruz, CA, USA; 1:3000) overnight at 4 with gentle shaking. The Mirogabalin web immune complex was detected with a 1:4000 dilution HRP conjugated anti rabbit secondary antibody. Gapdh (Catalog#: sc-25778, Santa Cruz Bio.) was detected as the loading control. NAD/NADH assays were performed using an AmpliteTM fluorimetric total NAD/NADH assay kit (Catalog#: 15257, AAT Bioquest, Sunnyvale, CA, USA) according to the manufacturer’s instruction. 10 protein for each sample was applied for the assay.Chromatin immunoprecipitation (CHIP) assay and luciferase reporter assaylysine 9 (K9). Immunoprecipitate.
