Celllytic lysis buffer

The enzymatic activities of GCase in peripheral blood lymphocytes was determined with the synthetic fluorogenic substrate 4-methyl umbelliferyl-b-D-glucopyranoside (4-MUG), according to commonly used methods (Papagiannakis et al., 2015 (link); Kim et al., 2016 (link)). PBL pellets (5 × 10⁶ cells/each) were lysed for 30 min on ice in CellLytic lysis buffer (Sigma). A reaction mix was prepared (0.15 M citrate/phosphate buffer, pH 5.9; sodium taurocholate and 1 nM 4-MUG) and added to each sample and blank well. Samples were incubated 3 h at 37°C on a shaker. The standard 1 nM 4-Methylumbelliferone (Sigma) was added before reading and the reaction was stopped with 1 M Glycine, pH 10.4. Each sample was measured independently six-fold. Fluorescence (excitation: 355 nm; emission: 460 nm) was measured using a microplate reader (Molecular Devices) and the final result is reported as nmol of substrate per hour per microgram protein.

Cells were grown to confluence in 6 cm petri dishes, differentiated for 9 days (peak adipogenesis in the second AIM induction) and 21 days (endpoint of adipogenic induction) using the adipogenic protocol with and without NAM treatment and harvested in CellLytic lysis buffer (Sigma-Aldrich) with 0.01% protease inhibitor (Sigma-Aldrich) added. Cell lysate was sonicated and centrifuged; the supernatant was flash frozen and stored at -80°C until assay.
SIRT1 protein was extracted from cell supernatant using immunoprecipitation. Briefly, a 1:50 dilution of SIRT1 antibody (Abcam) was added to each sample of cell supernatant and incubated overnight at 4°C with agitation. The antibody-supernatant mixture was added to a 50% slurry of protein A agrose bead (Cell Signaling) and incubated for 3 hours at 4°C with agitation. SIRT1 protein activity was then measured by fluorometric assay at 350 nm (Abcam) according to the manufacturer’s protocol for quantification of SIRT1 activity.
Total SIRT1 protein was measured simultaneously by Simple Western size-based protein assay (WES, ProteinSimple, Santa Clara, CA) following manufacturer’s protocol. Results from WES were analyzed using ProteinSimple Compass software. SIRT1 activity in each cell set was normalized to the cell set’s total SIRT1 protein as measured by WES.

HRMEC were seeded in 6-well plates and grown to 75% confluence, before being transfected with control, NFATc1-, NFATc2-, NFATc3-, or NFATc4-directed siRNA as described above. Twelve hrs post-transfection, cells were switched to 2% medium for 12 hrs, before being placed in 0.5% medium for an additional 6 hrs. Cells were then stimulated with either 0.5% medium or 0.5% medium plus 1 ng/ml TNFα for 6 hrs. After treatment, culture medium was collected and assayed for secreted CXCL10, CXCL11, MCP-1, and IL-6 protein concentrations using protein specific colorimetric sandwich ELISA kits (R&D Systems; Minneapolis, MN). Cells were washed with cold PBS and lysed using CellLytic lysis buffer (Sigma Aldrich), and the concentration of cell lysates was determined using a bicinchoninic acid assay (Pierce; Rockford, IL). Secreted protein concentrations were normalized to total protein of corresponding cell lysates and reported as pg/mg of total cellular protein.