Bovine serum albumin (bsa)

Cells were seeded on coverslips coated with 0.1% gelatin in 6-well plates, incubated under hypoxic/normoxic conditions for 4 days, and then fixed with 3.7% paraformaldehyde (Junsei Chemical, Tokyo, Japan). After permeabilization in 0.5% Triton X-100 (VWR Life Science, Radnor, PA, USA)/phosphate-buffered saline (PBS-T) and several washes with PBS, the cells were incubated with an anti-E-cadherin antibody (Cell Signaling) diluted in 1% bovine serum albumin (BSA)(MP Biomedicals, Santa Ana, CA, USA)/PBS-T for 1 h at room temperature. Afterward, the cells were incubated with Alexa Fluor 555 donkey anti-rabbit IgG (Life Technologies, Carlsbad, CA, USA) and mounted in Vectashield containing DAPI (Vector Laboratories, Burlingame, CA, USA). Images were acquired at 200× magnification with an LSM710 confocal microscope (Carl Zeiss, Jena, Germany).

The kidney samples were fixed in 4% buffered paraformaldehyde, embedded in paraffin, then 5 μm sections were cut for hematoxylin and eosin (H&E) staining, terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick-end labeling (TUNEL) assay and immunohistochemistry staining. The tissue sections were dewaxed and rehydrated, stained with hematoxylin and eosin, then observed under a light microscope (Nikon, Japan).
The TUNEL assay was performed as described previously (Liang et al., 2016 (link)). Briefly, sections were blocked with 5% bovine serum albumin (BSA, MPbio, California, United States) for 1 h at room temperature, then incubated with reagents from the TUNEL system kit, according to the manufacturer’s instructions. Green-labeled TUNEL-positive cells were observed under a fluorescence microscope at ×100 magnification.
Sections after dewaxed and rehydrated, were blocked by 5% BSA for 60 min. After blocking, the sections were incubated with primary antibodies against BIP at 4 °C overnight and then were incubated with anti-rabbit second antibodies (Boster, Wuhan, China) at room temperature for 1 h. The nuclei were stained with hematoxylin. The quantification of all raw images was performed using Image-Pro Plus 5.0 software.

After the indicated treatment, cells in 6-well plates were lysed on ice for 15 min by Radio Immunoprecipitation Assay (RIPA) Lysis Buffer (Beyotime Biotechnology, Beijing, China) containing PMSF, protease inhibitor (cOmplete, Roche, Basel, Switzerland), and phosphatase inhibitor (PhosSTOP, Roche, Basel, Switzerland). Cell lysates were centrifuged at 13,000× g for 15 min at 4 °C. Proteins in the supernatant were collected and measured by Micro BCA Protein Assay Kit (Thermo Fisher Scientific, Waltham, MA, USA). An equal amount of total protein was separated on 10% SDS-polyacrylamide gel before transferring onto equilibrated PVDF membranes. The membranes were blocked with 5% (w/v) Bovine serum albumin (BSA, MP Biomedicals, Santa Ana, CA, USA). After that, incubation of specific primary antibodies was carried out overnight at 4 °C. After washing for 3 times with TBST, the membrane was incubated with appropriate secondary antibodies at room temperature for 1 h. Signals were detected by ChemiDoc Touch Imaging System (Bio-Rad, Hercules, CA, USA) using Pierce™ ECL Western Blotting Substrate (Thermo Fischer Scientific, Waltham, MA, USA). Densitometric scanning of the desired band was corrected by β-actin to determine the changed level of specific protein. Each immunoblotting experiment was carried out for at least two times.

Isolation of cerebral arteries and subsequent enzymatic VSMCs isolation were carried out as previously described [9 (link),31 (link)]. Briefly, the brain tissue was carefully removed and placed in 4 °C physiological salt solution (PSS) containing (in mM) 137 NaCl, 5.6 KCl, 1MgCl₂, 0.42 Na₂HPO₄, 0.44 NaH₂PO₄, 4.2 NaHCO₃, and 10HEPES, equilibrated with 95% O₂ and 5% CO₂ at pH adjusted to 7.4 with NaOH. The cerebral arteries, including superior, middle, and basilar arteries, with the circle of Willis were dissected out and then harvested. Cerebral arteries were cut into 1–2 mm length and digested for 18 min at 37 °C with solution containing 4 mg/mL papain (Biochrom, Berlin, Germany), 2 mg/mL dithioerythritol (Amresco, St. Louis, MO, USA), 1 mg/mL bovine serum albumin (BSA) (MP Biomedicals, Illkirch, France), and 5 mM taurine in PSS. Following, the segments were transferred to enzyme-free PSS containing 1 mg/mL BSA and 5 mM taurine at room temperature for 10 min and triturated with a flame-polished pipette to disperse VSMCs. Isolated VSMCs were suspended in Ca²⁺-free PSS containing 1 mg/mL BSA and 5 mM taurine and stored at 4 °C for use within 8 h.

The replication of recombinant viruses in primary CEK and TEK cells as well as indicated cell lines was compared using a multiplicity of infection (MOI) of 0.001. Viruses were incubated with the indicated cells in 12-well plates at 37 °C and 5% CO₂ for 1 h (h). The virus inoculum was removed and cells were treated with citrate buffered (pH 3.0) saline (CBS) for 2 min (min) to inactivate extracellular virions. Afterwards, cells were washed twice with phosphate buffer saline (PBS). Finally, cells (except MDCK-HAT and MDCK-TMPRSS2) were covered with minimal essential medium (MEM) containing 2.8% bovine serum albumin (BSA) (MP Biomedicals, USA) and with or without 2 µg/mL TPCK-treated trypsin (Sigma-Aldrich, USA) and incubated at 37 °C and 5% CO₂. MDCK-HAT and MDCK-TMPRSS2 were covered with MEM containing BSA and 0.2 µg/ml Doxycycline (Sigma-Aldrich, USA) as described^{29 (link)}. Cells were harvested at the indicated time points and stored at − 70 °C until use. The replication kinetics were conducted in duplicates and repeated two times for each type of cells. Virus progeny was titrated by plaque test as described below. The results are expressed as mean and standard deviation of all replicates as log₁₀ plaque forming unit per ml (Log₁₀ pfu/ml).