Protease inhibitor

Fluorogenic caspase 3 substrate Ac-YVAD-AMC (Peptide Institute Inc., Osaka, Japan) was used to identify caspase activation. An aliquot of 1 × 10⁶ macrophages were harvested in a reaction buffer containing 100 mM HEPES, 10% (w/v) sucrose, 0.1% (w/v) 3-[(3-cholamidopropyl)-dimethylammonio]-1-propanesulfonate (CHAPS), 10 mM DTT, protease inhibitors (Biovision, California, USA) and triton 1%. After harvesting, caspase activity was detected by a fluorogenic assay as described by Thornberry [20 (link)]. A total of 200 µg of cell homogenates were placed in 96 dark ELISA plates with 25 µM of fluorogenic substrate. Emission fluorescence in the 380/430–460 nm range was determined for 20 min using the Synergy HT Bio Tek microplate reader. Caspase activity was expressed as, fluorescence produced per minute during 15 min per 200 µg of protein and presented as mean ± standard deviation from two independent experiments. Differences among positive control and cellular lysates from M. bovis-infected macrophages at different time points were tested for statistical significance by one-way ANOVA. All tests were performed using SPSS18 software for windows. A p value equal or less than 0.05 was considered to be statistically significant.

Lung RNA was isolated (RNeasy #74104; Qiagen, Hilden, Germany), quantified (Thermofisher nanodrop 2100), and quality assessed (Agilent Bioanalyzer). For protein analysis, lung was homogenized using 50 mM TrisHCl, 150 nM NaCl, NP-40-1%; sodium desoxycholate – 0.5%W/V; SDS-0.1%, radio immunoprecipitation solution (RIPA buffer #RB4476 Biobasic; Ontario, Canada) in 1:100 ratio supplemented with protease inhibitors (# ab6562 Abcam, Cambridge, UK) and centrifuged (10,000×g, 15 min, 4 °C). The supernatant was aspirated. Total protein content was determined spectrophotometrically (620 nm Protein Assay Dye Reagent # 500–0006; BioRad, USA).

Western blot analysis was carried out as described previously.17 Briefly, total proteins were isolated from normal or OA chondrocytes by RIPA buffer (Beyotime Biotechnology, Beijing, China) containing protease inhibitors (Abcam, Cambridge, UK) to obtain whole cell extracts. Membranes were incubated with primary antibodies against RUNX2 (1:1000 dilution; Cell Signalling Technology, Boston, USA); β‐actin (1:3000; Cell Signalling Technology); HDAC2, HDAC8, COL2A1 and MMP13 (1:1000; Abcam); aggrecan and SOX9 (1:2000; Millipore, Bedford, USA). β‐actin was used as an internal control. Immunohistochemical analysis was performed as described previously.17 Cartilage tissue sections were blocked in PBS plus 0.025% Tween 20 with 10% foetal bovine serum, followed by incubation with rabbit anti‐human HDAC2/8 antibody (1:200; Abcam).

Cellular proteins were extracted, and western blotting was performed as previously reported (Mao et al., 2017 (link)). Briefly, total proteins were isolated from BMSCs with radioimmunoprecipitation assay (RIPA) buffer (Beyotime Biotechnology, Beijing, China) containing protease inhibitors (Abcam, Cambridge, United Kingdom). Membranes were incubated at 4°C overnight with primary antibodies against RUNX2 (1:2,000; Cell Signaling Technology, Danvers, MA, United States), OCN, OPN, and SORBS1 (1:1,000; Proteintech, Wuhan, China). GAPDH (1:3,000; Cell Signaling Technology, Danvers, MA, United States) was used as the internal control. Appropriate secondary antibodies (1:3,000; Cell Signaling Technology, Danvers, MA, United States) were incubated with the blots at 20–25°C for 1 h. After rinsing with Tris-buffered saline (TBS) containing 0.05% (w/v) Tween-20 (TBST), the signals were detected using an enhanced chemiluminescence (ECL) kit (Millipore, Darmstadt, Germany) and a chemiluminescence system (Bio-Rad Laboratories, Hercules, CA, United States) and analyzed with Image Lab v6.0 software.¹

Intact brain-tissue was dissected-out after rapid decapitation. Brains were placed in RNA later (Sigma, USA) for 24–48 h at 4°C. Various anatomical regions of the brain tissue were dissected under microscope. Thereafter, 10% (w/v) homogenates were prepared in buffer containing protease inhibitors (Abcam, USA). For analysis, samples were normalized for total protein content, levels of neurotransmitters (viz. serotonin, glutamate, dopamine), cytokines (viz. IFNγ, IL-17A) were estimated in tissue homogenates using commercially purchased kits from BioVision Inc., USA and ELISA based kits provided by Ebioscience, USA respectively. Observed levels were normalized to control (medium or homogenization buffer).