H4784

Samples were subjected to 5 serial dilutions, with the antibody titre resulting in an 80% reduction in the luciferase signal produced by control wells containing PsV and cells only estimated by interpolation. HPV antibody control reagents were included in each assay run60 alongside heparin (H-4784; Sigma-Aldrich) which was used as a positive inhibitor control: Median heparin concentration (μg/mL) against PsV HPV31 5.5 (IQR, 3.8 to 6.7; n = 6) and HPV35 3.1 (IQR, 2.5 to 3.3; n = 6); Median neutralisation titres of the positive antibody control reagent (high titre HPV16/18) against PsV HPV31 389 (IQR, 333 to 427; n = 12) and HPV35 46 (IQR, 42 to 53; n = 12). The negative antibody control reagent (HPV negative) had a titre of <40 in all assays (n = 24).

EV71-VP1_97R/L167G/E mixed viral population was pre-incubated with 300 μg/ml of soluble heparin sodium salt from porcine intestinal mucosa (H4784, Sigma-Aldrich, Merck, Switzerland) diluted in culture medium or with culture medium alone as control for 1 h at 37°C. Confluent monolayers of Vero, Caco-2, RD or SH-SY5Y cells were challenged with 50 μl of the pre-incubated virus-heparin or virus-alone mixture and virus binding and replication assays were performed.

The colorectal cell lines HT-29 (ATCC HTB-38) and SW620 (ATCC CCL-227) were used as control for several tests, as well as the lung cancer cell line A549 (ATCC CRM-CCL-185), the mammary cancer cell lines SKBR3 (ATCC HTB-30) and BC-M1 (an established breast cancer cell line from a single disseminated tumor cell in the bone marrow of a patient with non-metastatic breast cancer patient)^{37 (link)}. HT-29 and SKBR3 cells were maintained in Dulbecco’s Modified Eagle’s medium (DMEM), 10% fetal bovine serum (FBS), and 1% penicillin/streptomycin. A549 cells were maintained in DMEM supplemented with 10 mM HEPES, 10% FBS and 1% penicillin/streptomycin. SW620 cells were maintained in RPMI 1640 with L-glutamine and 10% FBS, and the colon CTC lines and BC-M1 cells in CTC culture medium. Endothelial cells were cultured in M199 (M4530, SIGMA ALDRICH, St Louis, USA) containing 0.4 ng/ml endothelial growth factors (βECGF; E1388, SIGMA ALDRICH), 20 µg/ml heparin (H4784, SIGMA ALDRICH, Saint-Louis, USA), 1% penicillin/streptomycin and 20% FBS (endothelial cell medium).

All of the blood samples were heparinized with 1000 UI/mL of sodium heparin (H4784, Sigma, St. Louis, MO, USA) in avian saline. After centrifugation at 1000× g for 20 min, the plasma was decanted and stored at −80 °C. Concentrations of MEL in plasma were measured by an enzyme-linked immunosorbent assay kit for anti-MEL (CEA908Ge, Uscn Life Science, Inc., Wuhan, China).
The cells were incubated with 1 μM of prazosin for 30 min followed by 250 pg/mL of MEL for 24 h, and 3-isobutyl-1-methylxanthin (IBMX 100 μM, T1713, Topscience, TX, USA) was added to the medium to prevent cAMP degradation before the cells were incubated for a further 30 min. The cells were collected and lysed with lysis buffer from the cAMP ELISA Kit (R&D Systems, Minneapolis, MN, USA). Further treatment of the samples was performed according to the manufacturer’s instructions.
The cells were incubated with 1 μM of prazosin or 10 μM of PD98059 for 30 min, followed by 250 pg/mL of MEL for 24 h. The supernatant was collected for IGF-I protein detection by the anti-chicken IGF-I ELISA kit (SEA050Ga, Uscn Life Science, Inc., Wuhan, China), according to the manufacturer’s protocols.

Quantification of mouse Flt1 protein levels was performed according to the manufacturer's instructions (R&D Systems, Quantikine MVR100). Briefly, MN explants of two E11.5 SCs were cultured in 250 μl starvation medium, either in absence or presence of 10 U ml⁻¹ heparin (H4784, Sigma-Aldrich) for 20 h. Total cellular protein of MN explants was extracted using ELISA-extraction buffer. MN-CM was directly subjected to the assay.

To evaluate the involvement of HSPG-dependent replication by different EV-A71 strains, HSPG-binding sites on the viruses were blocked with low molecular weight heparin (LMWH; H4784, Sigma Aldrich). EV-A71 strains, 10⁵ TCID50/100 μL, were pre-incubated with 5 mg/mL LMWH for 30 min at 37°C with 5% CO₂. Subsequently 100 μL or 600 μL of the virus/LMWH mixtures were, respectively, added to the apical or basolateral compartment of the Transwell® inserts. The monolayers were incubated for 2 h at 37°C with 5% CO₂ and subsequently washed three times with PBS. Fresh differentiation medium was added, and the monolayers were incubated for 10 min, after which the 0-h time point was collected, removing 100 μL from the apical and the basolateral side. At 24-, 48-, and 72-h post infection, a 100 μL sample was taken from the apical and the basolateral compartment and the collected volume was replaced with fresh differentiation medium.

After IP injection of an overdose of ketamine (300 mg/kg) and xylazine (60 mg/kg), the abdominal wall and thorax of the mice were opened to expose the heart. The mice were perfused at 100 ml/h using a multichannel syringe pump (ProSense B.V.) via the right ventricle using Krebs-Henseleit buffer (Sigma-Aldrich, K3753) containing 10 U/mL heparin (Sigma-Aldrich, H4784) to rinse away the blood.
Lung tissue was processed for several purposes, i.e., real-time quantitative PCR (RT-qPCR), nCounter digital transcriptomics profiling, flow cytometry-based immune-profiling, scanning electron microscopy, immunofluorescence microscopy and histopathological staining.