The labeled sperm cells were analyzed by flow cytometry

typical morphological features like multiple fat vacuoles was determined by scoring 100 cells from 20 different microscopic fields per culture and the number of adipocytes was expressed as an indicator of adipogenic differentiation of MSCs. A detailed description of the technique used for the following experiments has been previously published. Briefly, highdensity cultures were rinsed in PBS and the proteins extracted with lysis buffer, 150 mM NaCl, l% Triton X-100, 1 mM sodium orthovanadate, 50 mM sodium pyrophosphate, 100 mM sodium fluoride, 0.01% aprotinin, pepstatin A, leupeptin and 1 mM phenylmethylsulfonyl fluoride ) for 30 min on ice. After adjusting the total protein concentration, samples were separated by SDSPAGE under reducing conditions. For immunoprecipitation, the extracts were pre-cleared by incubating them first with 25 ml of either normal rabbit IgG-serum or normal mouse IgG-serum and Staphylococcus aureus cells, then with primary Cobicistat site antibodies diluted in wash buffer, 1 mM CaCl2, 1 mM MgCl2 and 1 mM PMSF) for 2 h at 4uC, and finally with S. aureus cells for 1 h at 4uC. Control immunoprecipitations were performed by incubating the samples with non-immune rabbit anti-mouse IgG alone. S. aureus cells were washed five times with wash buffer and once with 50 mM Tris-HCl and then boiled in SDS-PAGE sample buffer. Separated proteins were transferred to nitrocellulose membranes and incubated in blocking buffer skimmed milk powder in PBS/0.1% Tween 20) for 1 h at AT. Membranes were incubated overnight with the first antibody diluted in blocking buffer at 4uC on a shaker, washed three times with blocking buffer, and then incubated with the secondary antibody conjugated with alkaline phosphatase for 90 min at AT. Membranes were rinsed with blocking buffer and then washed three times in 0.1 M Tris containing 0.05 M MgCl2 and 0.1 M NaCl. Specific antigen-antibody complexes were rendered visible using nitro-blue tetrazolium and 5-bromo-4chloro-3-indoylphosphate as the substrates for alkaline phosphatase. Total protein concentration was determined according to the bicinchoninic acid system using bovine serum albumin as a standard. Specific binding was quantified by densitometry using “quantity one”. Runx2 acetylation assay Runx2 lysine acetylation was analyzed by immunoprecipitation of Runx2 followed by western blotting using acetyl-lysine antibodies. Cells were treated with 1 mM resveratrol for 4 hours and then exposed to 1, 10 and 100 mM nicotinamide for indicated times. Whole-cell extracts were prepared, immunoprecipitated with an anti- Runx2 antibody, and subjected to western blot analysis using an antiacetyl-lysine antibody. To confirm these observations, MSCs were treated with 1 mM resveratrol for 4 h and then transfected with specific Sirt-1 antisense or sense oligonucleotides at 1 mM end concentration for 24 h. After 24 h of incubation transfection media were replaced with the regular Immunofluorescence analysis of Sirt-1 The effect of specific Sirt-1 antisense or sense on the Sirt-1 expression was investigated by an immunofluorescence method as previously described in detail. Briefly, the MSCs were cultured in 4-well glass plates and incubated for 24 h. Serumstarved cells were treated with 1 mM end concentration of antisense or sense for 24 hours in serum-starved medium. Glass plates were rinsed three-times in Hanks solution before methanol Resveratrol Promotes Osteogenesis of MSCs culture medium or osteogenic induction medium