Protein assay reagent

The exosome-producing medium was prepared to remove residual exosomes from FBS as referred [35 ] with modification. Generally, 50% (v/v) FBS in DMEM medium was centrifuged at 2,000 × g for 10 min, and then centrifuged at 100,000 × g (Beckman Optima L90-K with 90Ti rotor; Beckman Coulter Taiwan Inc., Taipei, Taiwan) for 16 hr at 4° C. The supernatant were collected and diluted into 10% (v/v) FBS by serum-free DMEM, and were sterile through 0.22 μm filter. For production of hepatoma-derived exosomes, 1 × 10⁶ Hep3B or Huh7 cells were plated in culture medium overnight, and were replaced into exosome-producing medium for successive culture for 2 days. Exosome-containing medium (100 mL) were collected and exosomes were isolated by Total Exosome Isolation kit (Life Technologies, Grand Island, NY, USA) according to suggested protocol. The pellets containing secreted exosomes were further washed by DEPC-treated PBS, centrifuged at 100,000 × g for 60 min (Beckman Optima MAX-E with TLA-120.2 rotor), and repeated twice to remove residual serum protein. The protein content in the exosome solution was determined by protein assay reagent (Thermo Fisher Scientific Inc., Pittsburgh, PA, USA).

The following antibodies were used for western blot analyses: anti–Cyclin D1 (Abcam, cat. # ab16663); anti‐p21 (Cell Signaling, cat. # 2947); anti‐PARP (Cell Signaling, cat. # 9532); anti–caspase‐3 (Cell Signaling, cat. # 9664 and 9665); anti–actin, HRP coupled (Sigma‐Aldrich, cat. # A3854); anti‐TLR7 (Proteintech cat. #17232‐1‐AP).
For protein extraction, cells were collected together with medium and centrifuged at 1600 rpm at 4°C for 5 minutes. Pellets were washed twice with ice‐cold PBS and then resuspended in 200 ml lysis buffer (PBS containing protease inhibitors (Protease Arrest, GBiosciences) and phosphatase inhibitors (PMSF 1 mmol/L, EDTA 0.5 mmol/L, sodium pyrophosphate 25 mmol/L, sodium orthovanadate, 10 mmol/L, sodium fluoride 50 mmol/L)). Cells were sonicated (LabSonic, BBraun) and protein content was assessed using Protein Assay Reagent (Thermo Scientific). For Western blotting, 15 mg proteins were electrophoresed on SDS‐polyacrylamide gels and electrophoretically transferred onto nitrocellulose membranes (Optitran, GE Healthcare Life Sciences). Membranes were blocked in 5% nonfat dry milk in TBST (10 mmol/L Tris–HCl, pH 7.6, 100 mmol/L NaCl, 0.1% Tween 20) for 2 hours at room temperature and then probed with appropriate antibodies.

For histologic analysis, lungs of animals were flushed with 1ml of 10% formalin, and sections were then removed and fixed in 10% formalin for 24 hours prior to being embedded in paraffin. Lung sections were then stained with hematoxylin-eosin and examined by a pathologist (ABF) blinded to sample identity.
Myeloperoxidase (MPO) activity was also assessed in BAL fluid. The trachea was irrigated with 1 ml of PBS, and fluid was then withdrawn and centrifuged at 10,000 RPM for 10 minutes. Substrate buffer containing 0.0005% hydrogen peroxide and O-dianisidine was added to the supernatant, and MPO activity was assayed over 6 minutes at wavelength 460 (BioTek Synergy HT, Winooski, VT). MPO activity was calculated as optical density/minute per μl of BAL fluid.
Lung fluid protein concentration was measured by analyzing samples treated with protein assay reagent (Thermo Scientific, Rockford, IL) at 660 nm in conjunction with a standard curve of bovine serum albumin.

Protein was assayed using a modified Bradford assay. Cell lysate and DOSCAT were diluted 1:50 and 1:100 in Milli-Q (18 Ω) water and 100 μL of each sample added to a microtitre plate in duplicate followed by the addition of 200 μL protein assay reagent (Thermo Fisher Scientific, Cramlington, UK). The plate was read at 600 nm on a microplate reader (Multiscan) using Ascent software (Thermo Fisher Scientific) and protein concentrations interpolated from a BSA standard curve.

RIPA buffer was prepared by adding 20 µL of 50× EDTA-Free protease inhibitor cocktail (Sigma-Aldrich), and 100 µL PhosStop phosphatase inhibitor (Sigma-Aldrich) to 880 µL RIPA buffer (Boston Bioproducts). Mammospheres were pelleted using the same procedure described in the “RNA Extraction and Purification” section, and RIPA buffer plus protease and phosphatase inhibitor were used to resuspend and lyse the pellet. After incubating the cell lysate on ice for 30 min, the lysate was spun at 15,000× g for 20 min; supernatants were collected, and the pellet discarded. The protein in the supernatant was quantified using the Bio-Rad Protein Assay Reagent and NanoDrop One^© (Thermo Fisher).

Proteasome extraction from cells was carried out as described previously [47 (link)–50 (link)]. Briefly, cells (1×10⁷) were harvested, resuspended in 1 ml ATP/DTT lysis buffer [10mM Tris-HCl (pH 8), 5mM ATP, 0.5mM DTT, 5mM MgCl₂], and incubated on ice for 10 min. This was followed by sonication for 15 s. Lysates were centrifuged at 500 × g for 10 min at 4°C. The resulting supernatant containing proteasomes was stable at −80°C for ≥ 1 month with the addition of 20% (v/v) glycerol. Protein concentrations of proteasome extractions from mice and cells were measured using a BCA (bicinchoninic acid) protein assay reagent (Thermo Fisher Scientific Inc. Waltham, MA, USA) with bovine serum albumin as a standard. The fluorogenic substrates Succ-LLVY-AMC, Z-ARR-AMC, and Z-LLE-AMC were used to measure chymotrypsin-like (CT-L), trypsin-like (T-L), and peptidylglutamyl peptide hydrolyzing (PGPH) proteasome activities. Assays were carried out in 50μg, 50mM EDTA and 50 μM fluorogenic substrates in a total volume of 200 μl of ATP/DTT lysis buffer at 37°C°C. The fluorescent rate was determined using a Synergy HT (Bio-TEK Instruments Inc. Winooski, VT, USA) at an excitation wavelength of 395 nm and emission wavelength of 460 nm.

Cells were plated in 6-well culture plates the day before treatment/infection and harvested in lysis buffer containing 1% NP-40, 150 mM NaCl, 50 mM Tris-HCl pH 8.0 and a protease inhibitor cocktail (Thermo Fisher Scientific). After removal of cellular debris by centrifugation, total protein concentration was measured at 660nm using protein assay reagent (Thermo Fisher Scientific) and 25 μg of total protein were separated in a precast 12% mini-PROTEAN TGX gel (Bio-Rad) and transferred to a nitrocellulose membrane (Bio-Rad). The membrane was incubated with a rabbit polyclonal antibody to human OPN (Abcam, ab181440) and a mouse monoclonal antibody to β-actin (Sigma-Aldrich); proteins were detected by incubating with a secondary anti-mouse IgG-HRP and/or anti-rabbit IgG-HRP, followed by the ECL reagent kit (Pierce). Images were captured using a ChemiDoc XRS+ imaging system (Bio-Rad).