Protein assay

To obtain extracellular vesicles released from SH-SY5Y, SH-SY5Y-NTRK1, and SH-SY5Y-NTRK2 cells, cells were cultured for 9 h in RPMI medium supplemented with 10% extracellular vesicle-depleted fetal bovine serum (FCS), 5% penicillin-streptomycin (Pen Strep, 10,000 U/mL, Life Technologies), and 1% L-Glutamine (L-Glutamine, 200 mM, Life Technologies). Conditioned media was subjected to ultracentrifugation at 10,000 × g in a fixed angle Type 45 Ti rotor (Beckman Coulter) for 30 min in order to remove membrane patches, followed by a further ultracentrifugation step at 120,000 × g for 120 min at 4°C using a swinging bucket SW 40 Ti rotor (Beckman Coulter). Pelleted TEVs were resuspended in 0.9% NaCl and stored at −20°C until usage. The obtained TEV fractions were characterized by (i) SDS-PAGE and western blotting to verify typical extracellular vesicle marker expression (CD81, TSG101, syntenin) and the absence of intracellular proteins or endosomes (calnexin) according to consensus requirements defining extracellular vesicles (28 (link)), (ii) nano-particle tracking analysis using ZetaView analyses (Particle Metrix, Diessen, Germany) to define size and particle concentration (29 (link)) and (iii) protein assay (Thermo Scientific, Darmstadt, Germany) to define protein concentration.

Western blot assay was performed as previously described^{20 (link)}. Tissue or cell extracts were prepared using a dounce homogenizer in cold RIPA buffer supplemented with cocktail. The homogenates were centrifuged at 12,000 rpm for 15 min, and the protein concentrations were determined using a protein assay from Thermo. The protein lysates were separated by SDS-PAGE and electrotransferred onto a nitrocellulose membrane. The membrane was scanned using the Image Lab statistical software (Bio-Rad). Antibodies against PAR (Trevigen, 4335-MC-100), PARP1 (Trevigen, 4338-MC-50), p-γH2AX (Ser139) (Trevigen, 4418-APC-020), Gabarapl1 (Abcam, ab86497), ATG12 (Abcam, ab109491), P62 (Cell signaling Technology, 39749), LC3 (Cell Signaling Technology CST, 3868), p-P53 (Ser15) (Cell signaling technology, 9284), p-ATM (Ser1981) (Thermo Fisher MA1-2020),FoxO3a (Cell signaling technology, 12829), pFoxO3a (Cell signaling technology, 9466), β-actin (Cell signaling technology, 3700), PGC1α (Abcam, ab54481), TFAM (Abcam, ab131607), NRF1 (Abcam, ab175932), Lamin B1 (Cell signaling technology,13435), α-tubulin (Cell signaling technology,3873) were used as primary antibodies.

Cells were lysed in RIPA buffer. The protein concentration of each sample was determined using a Protein Assay (Thermo Scientific, 23225). Protein samples (25 μg) were loaded and separated on 10% SDS–PAGE gels and then transferred to polyvinylidene fluoride (PVDF) blotting membranes. PVDF membranes were blocked in TBST buffer containing 5% nonfat dry milk for 1 h. Rabbit anti-Chi3l1 (Cell Signaling, 47066S), rabbit anti-STAT3 (Cell Signaling, 12640), rabbit anti-p-STAT3 (Cell Signaling, 9145), and anti-GAPDH (Cell Signaling, 5174s) antibodies were incubated overnight at 4°C. Afterward, the mixture was incubated with HRP-conjugated secondary antibodies in blocking solution for 1 h at room temperature. Finally, enhanced chemiluminescence substrate (Thermo Scientific, 34578) was added to the membranes, and the proteins were assayed according to the manufacturer's instructions.

PMA-differentiated U937 MΦ were washed in ice-cold phosphate buffered saline (PBS) and lysed using RIPA (radioimmuniprecipitation assay) buffer pH 7.5 (Cell Signaling Technology Europa, Leiden, The Netherlands), containing protease/phosphatase inhibitor cocktail (Cell Signaling Technology, Boston, USA). The protein concentrations were determined spectrophotometrically using the Pierce BCA (bicinchoninic acid) Protein Assay (Thermo Scientific, Rockford, USA). Proteins were loaded on NuPAGE® Novex® 4–12% Bis-Tris Gels, pre-cast polyacrylamide gels (Life Technologies GmbH, Darmstadt Germany). Proteins were transferred onto 0.45 μm nitrocellulose membranes [Millipore (Billerica, MA, USA)]. Primary Antibodies (see Additional file 1) were added and incubated overnight at 4 °C in blocking buffer (5% milk). Membranes were incubated with enhanced ECL-anti-goat IgG-POD antibody, ECL-anti-mouse IgG-POD antibody or ECL-anti-rabbit IgG-POD antibody. The peroxidase reaction was visualized by AceGlow chemiluminescence substrate (Peqlab, Erlangen, Germany) and documented by the Fusion-SL Advance™ imaging system (Peqlab) according to the instruction manual. The intensity of the specific western blot bands was quantified using the software ImageJ from the National Institutes of Health (Bethesda, USA).

The concentration of the purified M2e-NP and mM2e-NP proteins was determined by Pierce BCA (bicinchoninic acid) Protein Assay (Thermofisher, USA). The target protein (0–1500 ng) was coated on an ELISA plate overnight at 4 °C. Next, the protein solution was decanted and the wells were washed 3 times with TBS-T buffer. Later, the wells were blocked with 1% (v/v) blocking solution at room temperature for 2 h. The solution was removed and the wells were washed 3 times with TBS-T. Then, 150 μL of primary antibody [mouse monoclonal anti-M2e (1:2500; Abcam, Cambridge, UK) or mouse monoclonal anti-NP (1:3000; Abcam, UK)] were added into the wells, and the plate was incubated on a shaker at room temperature for 1 h. After an hour, the antibody solution was discarded, the wells were washed as previously described. About 150 µL of HRP-conjugated goat anti-mouse IgG was added into the wells and the plate was incubated on a shaker at room temperature for 1 h. Upon the incubation, the wells were emptied, and washed 3 times with TBS-T. For color development, 50 µL of 3,3′,5,5′-tetramethylbenzidine (TMB) substrate was added into each well, and the enzymatic reaction took place at room temperature for 15 min. The plate was read at 605 nm. The OD readings were normalized using the blank controls, and the curves were plotted.

To avoid cellular damage, liver mitochondrial extraction was carried out within 1-2 h. Each group’s liver samples were pooled, weighed, homogenized, and washed in homogenate buffer. In a glass Potter-Elvehjem tissue grinder, liver specimens were homogenized with an appropriate volume of the homogenate buffer (4 mL homogenate buffer/1 g of the liver specimen). Several up and down strokes were performed with a motor-driven Teflon pestle at 600 rpm during this setup. The homogenates were then centrifuged at 1000 × g for 5 min at 4°C. The supernatants were collected and centrifuged at 15,000 × g for 2 min at 4°C. The mitochondrial pellets were collected and washed in homogenate buffer several times. The pellets were resuspended in an ice-cold final equilibrated buffer (250 mM sucrose, 5 mM KH₂PO₄, 10 mM Tris-HCl, and 2 mg/mL bovine serum albumin [BSA]; pH 7.2). Subsequently, 200 μL of the resuspended pellet was fixed in 2.5% glutaraldehyde in 0.1 M sucrose phosphate buffer for electron microscopy analysis. The protein content of mitochondria was determined using a spectrophotometer (NanoDrop-1000, Thermo Scientific, Wilmington, DE, USA) and a protein assay (Bio-Rad^®, Hercules, CA, USA).