Sh 1000

Cells were cultured in the same conditions as described in the ALP activity staining subsection above, then washed with PBS and homogenized with 1% Nonidet P-40 (50 μL) under sonication on ice. Cell lysates (10 μL) were added to 50 μL of 0.2 mol/L Tris–HCl buffer (pH 9.5) containing 1 mmol/L MgCl₂ and 12.5 mmol/L disodium p-nitrophenyl phosphate (Wako Pure Chemical Industries). After incubation for 15 minutes at 37 °C, reactions were terminated by addition of 50 μL of 0.5 mol/L NaOH and absorbance of the reaction mixture at 405 nm was read using a micro-plate reader (SH-1000, Corona Electric, Ibaraki, Japan). The increase in absorbance in after 15 minutes was divided by the amount of cellular protein and the obtained value was used to express the specific activity of ALP.

ELISA was performed to test the neutralization function of the A7 Fab. Recombinant ACE2 (ab151852, Abcam, Cambridge, UK) was immobilized on a microplate at a concentration of 2 µg/mL. After overnight incubation at 4 °C, the solution was removed and the microplate was blocked with 20% (v/v) ImmunoBlock (KAC, Hyogo, Japan) in PBST. After 2 h, the plates were washed again and 10 nM biotin-SARS-CoV-2 S1 protein (40591-V08H-B, Sino Biological Inc., Beijing, China) with purified A7 Fab antibody fragment (100 µL, at concentrations of 0.1–5000 nM, pre-incubated for 1 h at room temperature) was added into each well, while only 10 nM biotin-S1 protein was added in the control group. After incubation for 2 h, the plate was washed three times with PBST, followed by the addition of 100 µL Neutravidin-HRP (1:1000 dilution in PBST containing 5% ImmunoBlock). After 1 h of incubation and washing, 100 µL of substrate solution (0.2 mg/mL TMBZ and 30 mM H₂O₂ in 100 mM sodium acetate, pH 6.0) was added to develop the assay, and the absorbance at 450 nm and 655 nm was measured by a microplate reader SH-1000 (Corona Electric, Ibaraki, Japan) after terminating the reaction with 50 µL of 10% H₂SO₄. The inhibition constant K_i was estimated according to the Eq. (1). $${\text{IC}}_{50}=K_{\text{i}}\times (1+\frac{[\text{S}1]}{K_{\text{D}(\text{ACE2 against S1})}})$$

The HUEhT-1 cells were seeded at 1 × 10⁴ cells/well in 96-well culture plates with 100 µL medium and incubated overnight at 37 °C. Exosome samples were applied to the cells. After 48 h, 10 µL of an MTT labeling reagent in an MTT Cell Count Kit (Cat #23506, Nacalai Tesque) was added to each well, and the cells were incubated for 4 h at 37 °C. The 100-µL solubilization buffer in the kit was then added to each well, and the plates were incubated overnight at 37 °C. Absorbance was measured at 570 nm and 405 nm, as its reference wavelength, with a microplate reader (SH1000, Corona Electric).

GTPase activity of dynamin 2 was determined by monitoring release of free orthophosphate using malachite green assay (47 (link)). The malachite green reagent was prepared by mixing solution A (17 mg of Malachite Green Carbinol base dye (229105, Merck) in 20 ml 1 N HCl) and Solution B (0.5 g Ammonium molybdate (277908, Merck) in 7 ml 4 N HCl) with filling up to 50 ml by MilliQ water followed by filtration through 0.45 μm membrane (S-2504, KURABO). In the assay, 0.2 μM dynamin in the presence of BIN1 at different molar ratio was mixed with 1 mM GTP in GTPase reaction buffer (10 mM Hepes, 2 mM MgCl₂, 50 mM NaCl, pH 7.5) with or without 0.005 μg/μl lipid nanotubes and incubated for 5 min at 37 °C. After the reaction was stopped on ice for 10 min, 160 μl of malachite green reagent was added to the 40 μl of the reaction mix in 96-well plate (442404, Thermo Fisher Scientific). After 5 min shaking at 1200 rpm with Digital MicroPlate Genie Pulse (Scientific Industries, Inc), released orthophosphate was colorimetrically quantified by measuring OD 650 nm using a microplate reader (SH-1000, CORONA ELECTRIC).

Protein expression of gp91 phox, Cu/Zn-SOD, and LOX-1 in HUVECs was determined by western blot analysis. Briefly, HUVECs were homogenized and centrifuged to extract proteins. Protein concentrations were determined by BCA Protein Assay Kit (P0011, Beyotime Institute of Biotechnology, Nanjing, China) with a microplate reader (SH-1000, Corona Electric Co., Ltd, Japan) at OD 562 nm. Every 30 μg aliquot of protein was separated by a SDS-PAGE gel and transferred onto a nitrocellulose membrane. The transferred membrane was incubated overnight with specific polyclonal antibodies: anti-gp91 phox (1:500, Abcam, USA), anti-Cu/Zn-SOD (1:500, Novus Biologicals, USA), anti-LOX-1 (1:500, Abcam) and anti-β-actin (1:1000, Abcam). After washing three times, the blots were incubated with a corresponding secondary antibody and the probed protein was visualized by luminolchemiluminescenceBeyo ECL Plus (P0018, Beyotime Institute of Biotechnology, Nanjing, China) and finally detected by autoradiography exposure. The membrane was then re-blotted with β-actin antibody as a loading reference. Densitometry of the probed protein bands was analyzed for the aliquotsand normalized to corresponding β-actin, which was expressed as fold increases compared to the normal control group.

Blood ammonia was assayed using a commercial kit (Ammonia Test Wako, Wako, Osaka, Japan). Whole blood sample was used in the lactate and ammonia analyses. Blood for lactate analysis was from mouse’s tail by a razor. Lactate Pro 2 can measure lactate ∼0.3 µL whole blood. Blood for ammonia analysis was sampled from the heart about 1 mL. Blood sample (1.0 mL) was immediately mixed with deproteinizing reagent (4.0 mL) and centrifuged at 600 g for 5 min at 4°C. Supernatant (2.0 mL) was transferred to the new tube and added the color reagent solution A (2.0 mL). After well shake, added the color reagent solution B (1.0 mL) and the color reagent solution C (2.0 mL). These processes were kept at on ice condition. The sample was then incubated in the water bath at 37°C for 20 min. Absorbance was measured using the micro plate reader (SH-1000, CORONA ELECTRIC Co., Ibaraki, Japan). We used unperformed PT group’s blood samples as Pre (sedentary) and immediately after PT blood samples were used as Post.