Cell culture, virus infection and transfection

Human microglia cells (HMC3; Procell, China) and Vero cells were cultivated in Dulbecco’s Modified Eagles Medium (DMEM) containing 10% fetal bovine serum (FBS) and 100 U/ml penicillin-streptomycin at 37 °C with 5% CO2.

The CV-A10 (subgenotype C, GenBank NO. MN557275), which was isolated during an epidemic in Xiangyang, China, in 2017, were proliferated in Vero cells for this investigation. To explore the effect of CV-A10 on glial cells, HMC3 cells were plated into 6-well plates. Next day, CV-A10 was inoculated into cells at a multiplicity of infection (MOI) of 0.1 for 2 h at 37 °C. Subsequently, the virus inoculum was discard and the maintenance medium (i.e., DMEM containing 2% FBS) was replaced. Cells and supernatants were collected at the indicated time points.

In order to further investigate the biological role of MST1/2, the overexpression and knockdown vectors of MST1/2, as well as the corresponding negative control, were constructed, synthesized and purchased from Genepharma (Shanghai, China). These vectors were transfected into HMC3 cells using Lipofectamine 3000 (Invitrogen, USA) according to the standard procedure. Following a 24 h transfection, the cells were used for subsequent experiments.

Quantitative real-time polymerase chain reaction(qRT-PCR)

The quantitation of the number of copies of the CV-A10 genome was achieved by qRT-PCR. Briefly, total RNA was extracted using TIANamp Virus RNA Kit (TIANGEN, China) according to the manufacturer’s instructions. Subsequently, a commercial Coxsackievirus A10 RNA detection kit (TIANLONG, China) was used to detect the viral load with a GENTIER 96 equipment (TIANLONG, China). Meanwhile, the standards for CV-A10 with known concentrations were concurrently measured with the test samples. Finally, the viral load of CV-A10 in each sample was calculated according to the standard curve.

Titration of viruses

Viral titers were determined by 50% tissue culture infectious dose (TCID50) analysis on Vero cells using the Reed-Muench method. The supernatants of the infected culture were collected at indicated time and serially diluted in serum-free DMEM. Ten-fold diluted culture was adsorbed onto confluent Vero cells in a 96-well plate for 2 h at 37 °C. Later, the inoculum was removed, infected cells were washed with sterile phosphate-buffered saline (PBS) and cultured with in DMEM with 2% FBS for 3∼5 days.

Western blotting (WB)

Cells infected with CV-A10 for the indicated time were lysed in RIPA lysis buffer (Beyotime, China) with protease/phosphatase inhibitor Cocktail (Boster, China) on ice for 30 min. A BCA protein quantification kit (Beyotime, China) was used to determine the concentration of extracted proteins. 30 micrograms of proteins were resolved via 12% sodium dodecyl sulfate polyacrylamide gel electrophoresis (SDS-PAGE) and transferred to the polyvinylidene difluoride (PVDF) membrane. 5% nonfat milk was used for transferred PVDF membranes block for 1 h at room temperature, and then the membranes were incubated overnight at 4 °C with primary antibodies (1:1000 dilutions), including VP1 (GeneTex, China), p-MST1/2 (Affinity, USA), MST1/2 (Affinity, USA), p-LAST1/2 (Affinity, USA), LAST1/2 (Affinity, USA), p-YAP (Affinity, USA), YAP (Affinity, USA), TLR3 (Abclonal, China), TRIF, RIG-I (Abclonal, China), MDA5 (Abclonal, China), MAVS (Abcam, USA), TRAF3 (Abcam, USA), TBK1 (Abcam, USA), TLR7 (Abclonal, China), MyD88 (Abcam, USA), IRAK4 (Affinity, USA), IRAK1 (Affinity, USA), TRAF6 (Abcam, USA), TAK1 (Abcam, USA), NF-κB (CST, USA), IL-1β (CST, USA), IL-6 (CST, USA), IL-8 (CST, USA), TNF-α (CST, USA), IRF3 (Affinity, USA), IFN-β (Affinity, USA) and β-actin (Abbinke, China). After washed with TBST for three times, the membranes were probed with the respective horseradish peroxidase (HRP)-conjugated IgG secondary antibodies (1:10000 dilution; Abbinke, China). Finally, we examined protein signals through the enhanced chemiluminescence (ECL) system (Biosharp, China).

Flow cytometry assay for the examination of inflammatory cytokines

Twelve inflammatory cytokines, namely, TNF-α, IL-12, IL-4, IL-17, IL-8, IFN-γ, IL-10, IL-1β, IL-6, IL-2, IFN-α and IL-5, are common cellular inflammatory factors in human immunity and play an important role in human immune regulation. In this study, we used a commercial Bio-Plex cytokine assay (RAISECARE, China) for testing according to the product specification. In brief, 25 µl sample, 25 µl trapped microspheres and 25 µl experimental buffer were mixed and incubated for 2 h by slightly shocking, followed by 25 µl of SA-PE antibody added and incubated for 30 min. After cleaning with 100 µl washing buffer and centrifuging for 5 min at 1500rmp/min, 300 µl washing buffer was supplemented and determined on the flow cytometer. Finally, the concentrations of inflammatory cytokines were calculated by a LEGENDplex v8.0 software.

Immunofluorescence (IF) assay

For IF experiments, cells were seeded on poly-l-lysine coated coverslips. Following infection with CV-A10, cells were fixed with 4% paraformaldehyde and permeabilized with 0.1% Triton X-100 for 15 min at room temperature. Blocking is done with 2% bovine serum albumin (BSA; Biofroxx, China) for 1 h at room temperature prior to incubation with primary antibodies (1:100 dilution), including VP1, YAP, TBK1, IRAK1, NF-κB. Following thrice washes with PBS, the cells were further incubated with Alexa Fluor® 488-conjugated goat-anti-mouse IgG and Alexa Fluor® 594-conjugated goat-anti-rabbit IgG (1:300 dilution; CST, USA). After washing three times again, the coverslips were mounted on glass with anti-fade reagent which contained 4’,6-diamidino-2-phenylindole (DAPI; Beyotime, China). Fluorescent images were acquired on a confocal microscope (Leica, Germany).

Statistics

All data are presented as the means ± SD at least three independent experiments. Statistical analysis was conducted using Graph Pad Prism software. When two groups were compared, the student’s t-test was applied. For three or more group comparisons, one or two-way analysis of variance (ANOVA) was performed, as appropriate, followed by Tukey’s or Bonferroni’s post hoc test, as appropriate.