Cells culture and antibody
RD cells and Vero cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM） containing 10% fetal bovine serum (FBS, Gibco) and incubated in a 37℃ environment containing 5% carbon dioxide.
The EV71 VP1 antibody was purchased from Abnova, Taiwan, China; the β-tubulin antibody was purchased from Bioprimacy, Wuhan, China; the Caspase3 antibody was purchased from Proteintech, Wuhan, China; and the HA tag antibody was purchased from CST, USA.
Virus propagation and titration
The EV71 strain BrCr (VR-1775) was purchased from ATCC and was propagated in RD cells. The cells grow to 80%, and then get infected with the virus. After 80% of the cells developed a cytopathic efect (CPE) of viral infection, the cell maintenance supernatant (DMEM containing 1% FBS and 1% 100 μg/ml penicillin-streptomycin) was collected and subjected to three freeze–thaw cycles to lyse the cells. The virus titer was determined by the 50% tissue culture infectious dose (TCID50 assay).
Construction of HA tag VGLL4 recombination plasmid
To construct a plasmid for VGLL4 overexpression (pHA-VGLL4), a fragment of VGLL4 was cloned into the Kpn I and Xho I sites of the HA tag fusion expression vector using the targeting sequences HA-VGLL4-F (5’-cgcggtaccCTATTTATGAAGATGGACCTGTT-3’) and HA-VGLL4-R (5’-aatctcgagTTAGGAGACCACAGAGGGGGAGT-3’).
Transfection and expression of plasmids
RD cells were seeded in 6-well plate and cultured in DMEM containing 10% FBS at 37℃ in a 5% CO2 environment. When the degree of monolayer cell confuence reached 60-70%, plasmids were transfected into cells by using lipofectamine 2000 (Invitrigen, USA) according to the manufacturer’s instructions. The expression of plasmids were analysed by Western blot assay.
Western blot assay
PBS-washed cell pellets were lysed in 1×RIPA buffer (CST, USA). The samples were centrifuged at 12,000 rpm for 15 min to harvest the supernatant, which were resolved by 10% SDS-PAGE and transferred to nitrocellulose membranes (BioRad, USA). The membranes were blocked with 2% bovine serum albumin (BSA, Sigma-Aldrich, Germany) and then incubated with the appropriate primary antibody overnight at 4℃. The membranes were washed with 1×PBST and incubated with a 1:5000 dilution of anti-rabbit or anti-mouse horseradish-peroxidase-conjugated antibody for 1.5 h. Following incubation, the membranes were washed extensively with 1×PBST. Immunoreactive bands detected using ECL reagents (Advansta, USA) were developed with Image Lab (Bio-Rad, USA）.
Real-time quantitative RT-PCR
The cells were lysed by TransZol up and total RNA was isolated from cells as the instruction. The RNA was reversed by TransScript® One-Step gDNA Removal and cDNA Synthesis SuperMix and analyzed with the PerfectStartTM Green qPCR SuperMixt. All the reagents were purchased from Transgen biotech, Beijing, China. The mRNA expression of EV71 VP1 was detected with sense primer 5’-GCAGCCCAAAAGAACTTCAC-3’ and antisense primer 5’-ATTTCAGCA GCTTGGAGTGC- 3’ targeting a conserved region of the VP1 gene. The GAPDH mRNA was detected using sense primers 5’- GACAACTTTGGTATCGTGGAA-3’ and antisense primer 5’- CCAGGAAATGAGCTTGACA−3’. All the primers were synthesized from Sangon Biotech, Shanghai, China.
The virus was diluted from 10－1 to 10－8 and added to Vero cells in 96-well plates; the cells were cultured at 37℃ for 5 days and observed daily. The TCID50 values were measured by counting the cytopathic effect (CPE). Further calculations were conducted using the Reed-Muench method.
The cytotoxic effect was assayed by Cell Counting Kit (CCK) (Beyotime Biotechnology, Shanghai, China).