Cell Culture, transfection and viral infection
PK-15 cells were cultured in Dulbecco’s Modified EagleMedium DMEM-F12 (GIBCO) containing 10% (v/v) foetal cattle Serum, 100 μg/ml penicillin /streptomycin mixtures at 37 °C with 5% CO2.
The lncRNA smart silencers were synthesized by Ribobio (Guangzhou, China) and siRNA (siCCR1) were synthesized by GenePharma (Shanghai, China). The sequences of siCCR1s were as follow: siCCR1A 5’-UCAUUGGCCUGAUCGGCAATT-3’, the sequences of siCCR1B: 5’-GGCUCUAUUUCAUUGGCUUTT-3’, the sequences of siCCR1C: 5’-GCAAGUAUCUACGGCAGUUTT-3’, the sequences of siSP1: 5’-GCAACAUCAUUGCUGCUAUTT-3’, the sequences of NC (Negative control): 5’-UUCUCCGAACGUGUCACGUTT-3’. PK-15 cells were seeded in 6-well or 12-well plates and grown to approximately 50-60% confluence for transfection. The cells were transfected with 50 nmol siRNA or 100nmol lncRNA smart silencers using Lipofectamine 3000 reagent (Invitrogen, Carlsbad, USA) according to the manufacturer’s protocol. The cells were harvested at the indicated times.
The inhibitors were synthesized by Beyotime (China). AG490 (S1509, JAK inhibitor), LY294002 (S1737, PI3K inhibitor), SP600125 (S1876, JNK inhibitor), SB203580 (S1863, p38MAPK inhibitor), U0126 (S1901, MEK1/2 inhibitor), and ZK811752 (SD3699, CCR1 inhibitor)
The JEV strain SA14-14-2 (GenBank accession: AF315119.1) was propagated in BHK-21 cells according to the protocol of Yang (S. Yang et al., 2013). All infections were carried out by incubating the cells with virus at the MOI = 1, then the inoculum was removed, the cells were washed three times with PBS and fresh mediuma was added. The infection was performed and the infected PK-15 cells were maintained in DMEM supplemented with 2 % FBS without penicillin /streptomycin mixtures.
Plasmid
Full-length pig lncRNA-NONSUST006715.1 oligos was synthesis by Nucleic acid synthesizer, Full-length pig SP1 CDS was inserted into the NheI and XhoI sites of the pcDNA3.1(+) vector (Invitrogen).
Real-time quantitative PCR analysis
Primers for lncRNA-NONSUST006715.1, CCR1, SP1, NF1, GATA1 and CEBP-alpha were designed using the Primer 5 software; Primers for glyceraldehyde 3- phosphate dehydrogenase (GAPDH) were used as an internal control. Total RNA was extracted from cells using TRIzol® Reagent (Invitrogen) according to the manufacturer's protocol. The reverse transcription of total RNA (1 μg) was performed using a RevertAid™ RT Reagent Kit (RR036A, Takara) in a 20 μl reaction volume according to the manufacturer. Primer information for the Real-time quantitative PCR is also available in the Supplemental information (Table S1).
Western blot analysis
Cells were lysed with RIPA lysis buffer (P0013B, Beyotime, China) and 1 mM PMSF (ST506, Beyotime, China). Protein concentration of cell lysate was determined by the BCA method (Pierce, Rockford, USA). Ten micrograms of total protein per sample was loaded onto sodium dodecylsulphate polyacrylamide gel electrophoresis (SDS-PAGE) at 80 V for 3-4 h and transferred to PVDF membrane at 350mA for 90 min (Version8, Roche, USA) using an electro-blotting method. After incubating in blocking buffer (PBST with 1% (w/v) BSA (A7030, Sigma)) for 1 h, membranes were incubated with rabbit polyclonal antibody for NS3 (GTX125868, Genetex, USA), rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) at 4 °C for 12 h. After primary antibodies were used, the membranes were washed before Horseradish Peroxidase (HRP)-conjugated Goat anti-rabbit IgG second-antibody (sc-2030, Santa Cruz, USA) was added for 1 h at room temperature and washed again. The membranes were visualized with an ECL Western blot detection kit (NC15080, Thermo). The β-actin (#4970, Cell Signalling Technology, USA) protein level was also examined as an internal control. The chemiluminescence intensity of each protein band was quantified using the Image J, and then protein levels were normalized by the amount of β-actin protein.
Chromatin immunoprecipitation assay
Formaldehyde was added at a final concentration of 1% directly to media of PK-15 cells. Fixation proceeded at room temperature for 10 min and was stopped by the addition of glycine to a final concentration of 0.125M for 15 min. Cells were centrifuged and rinsed 3 times in cold PBS with 1mMPMSF. Then, cell nuclei were collected according to the manufacturer's protocol, SimpleChIP Enzymatic CHIP Kit (#9002, Cell Signalling Technology, USA). Samples were sonicated on ice with an Ultrasonics sonicator at setting 5 for six 10 s pulses to an average chromatin length of approximately 400 to 800 bp. For the immunoprecipitation, 2 μg rabbit polyclonal antibody for SP1 (ab13370, Abcam, USA) in a final volume of 500 μl immunoprecipitation (IP) buffer were added in combination to the nuclear sonicate. After the immunoprecipitation, the IP was eluted and the DNA was recovered. DNA obtained from IP samples were quantified by real-time PCR and normalized to input DNA control samples. Primer information for the ChIP assay is available in the Supplemental information (Table S1).
Statistics
Data are presented as means ± SEM. Significant differences were analyzed by Mann-Whitney test or one-way analysis of variance (ANOVA) using SPSS software (ver, 20.0, SPAA Inc, USA). P-values < 0.05 were considered to be statistically significant.