Protease inhibitor

Aliquots of 30 × 10⁴ MDCK II cells were plated on each well of a 6-well, 35 mm plate and the culture medium (DMEM) containing 100 μM NAR or AICAR, with a final concentration of 0.1% of d6-DMSO was added. The cells were harvested after 48 h of incubation under 5% CO₂ at 37 °C. Quantities of 10 μM LY294002 were added 3 h before being harvested. The cells were rinsed twice with DMEM medium without serum, and recovered with 500 μL of the cell lysis buffer (60 mM Tris, 5 M urea, 1 M thiourea, 1% protease inhibitor (Nacalai Tesque), 1% CHAPS, 1% TritonX-100, 1% dithiothreitol, pH 8.8). The cells were disrupted by mild sonication using Bioruptor (Cosmo Bio, Tokyo, Japan) for 5 min (duty cycle 50%). Each crude protein sample was analyzed by agar gel (ATTO) according to isoelectric point with a buffer (upper electrode solution: 0.2 M sodium hydroxide; lower electrode solution: 6 mM phosphoric acid), and the gel was fixed with trichloroacetic acid, washed with water and equilibrated with a buffer (0.5 M Tris-HCl (pH 6.8), 2% SDS, 0.001% bromophenol blue). The agar gel was then applied to the SDS-polyacrylamide gel and separated by molecular weight, followed by fixation, Coomassie Brilliant Blue staining, decolorization, and silver staining. Silver staining was performed using Silver Stain II Kit Wako, according to the manufacturer’s instruction.

HPMCs were transfected with miR-99a-5p or negative control miRNA for 24 h and lysed with a buffer composed of 50 mM Tris-HCl pH 7.5, 2% sodium deoxycholate, and protease inhibitor (Nacalai Tesque). Protein extracts were reduced, alkylated, and digested overnight. Samples were labeled with TMT 6-plex reagents (Thermo Fisher Scientific) and then mixed before sample fractionation and clean-up. Labeled samples were analyzed by liquid chromatography coupled with tandem mass spectrometry (LC-MS/MS) using a Q Exactive Mass Spectrometer (Thermo Fisher Scientific) equipped with an UltiMate 3000 Nano LC System (Thermo Fisher Scientific). Raw data were processed by Mascot v2.1 (Matrix Science, London, UK). Protein expression levels were compared between the two samples and pathway annotation for differentially expressed proteins was performed using DAVID 6.8 analysis (https://david.ncifcrf.gov).

To prepare whole cell extracts, cells were lysed with SDS sample buffer (50 mM Tris-HCl (pH 6.8), 10% glycerol and 1% SDS). For nuclear extracts, cells were lysed in buffer A (10 mM Tris-HCl (pH 8.0), 10 mM KCl, 0.1 mM EDTA, 1.5 mM MgCl₂), 1x protease inhibitor (Nacalai Tesque, Kyoto, Japan), 10 µM MG132 (Peptide Institute, Osaka, Japan) and 10% NP-40 (final concentration 2%). After centrifugation, the precipitated nuclei were lysed with SDS sample buffer. The protein concentrations of the cell extracts were measured using a bicinchoninic acid (BCA) kit (Thermo Fisher Scientific Inc. Rockford, IL, USA). Proteins were separated by sodium dodecyl sulfate-polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to PVDF membranes (Immobilon-P transfer membrane, Millipore, Billerica, MA, USA). The blots were incubated with the primary antibodies indicated on the figures and then with a horseradish peroxidase-conjugated secondary antibody (Thermo Fisher Scientific Inc. Rockford, IL, USA). The protein bands were visualized using enhanced chemiluminescence (GE Healthcare, Pittsburgh, PA, USA).

Cells were washed once with PBS and lysed in RIPA buffer consisting of 50 mM Tris (pH 8.0), 150 mM NaCl, 0.5% sodium deoxycholate, 0.1% SDS, and 1% Triton X-100 containing protease inhibitor (Nacalai Tesque, Kyoto, Japan) for 10 min on ice. The cell lysates were centrifuged at 17700 × g for 15 min at 4°C. The supernatants were mixed with an equal volume of 2× sample buffer consisting of 3% SDS, 10% glycerol, 100 mM Tris-HCl (pH 6.8), 0.1% bromophenol blue, and 10% 2-mercaptoethanol, and denatured at 95°C for 5 min. The cell lysates were separated by 12% SDS-PAGE and the proteins transferred onto PVDF membranes (Millipore). The membranes were blocked with 5% skim milk in TBS-T consisting of 0.1% Triton X-100, 25 mM Tris-HCl (pH 7.4), 132 mM NaCl, and 2.7 mM KCl, and incubated overnight at 4°C with the appropriate antibody. Bound antibodies were detected using HRP-conjugated secondary antibodies (Jackson ImmunoResearch, West Grove, PA, USA). The bands were visualized using the chemiluminescent HRP substrate (Millipore) and ChemiDoc XRS+ Imager (Bio-Rad, Hercules, USA), and the obtained images were analyzed using Image Lab Software (Bio-Rad).

Adherent cells cultured for 24 to 48 h were harvested on ice with a cell scraper. An additional culture of serum-starved cells was trypsinized and kept in suspension for 1 h, then collected in tubes. The cells were the incubated at 37°C for 15 and 30 min, and harvested (Cheng et al., 2014 (link)). The cell suspensions were centrifuged at 300 ×g at 4°C for 5 min and the supernatant was discarded. The cell pellets were washed with chilled phosphate-buffered saline (PBS) and lysed in radioimmunoprecipitation assay lysis buffer (Nacalai Tesque, Kyoto, Japan) or NP40 lysis buffer (Wako, Osaka, Japan) containing protease inhibitor (Nacalai Tesque) and phosphatase inhibitor (Nacalai Tesque) cocktails for 5 min on ice. The cell lysate was centrifuged (16000 ×g at 4°C for 15 min). The lysate [10–20 µg protein, as measured with a BCA Protein Assay kit (Thermo Fisher Scientific)] was then mixed with SDS sample buffer (Nacalai Tesque), separated by SDS-PAGE using pre-made 7.5% or 5–20% polyacrylamide gel plates (e-PAGEL, Atto, Tokyo, Japan), transferred to iBlot® 2 Transfer Stacks PVDF mini membranes using an iBlot® 2 Dry Blotting system (Thermo Fisher Scientific), and immunoblotted with specific antibodies at 1:1000 to 1:5000 dilution (Alanko et al., 2015 (link); Torisu et al., 2013 (link)).