Cell lines, viruses, plasmid, compounds and antibodies
HEK-293T (CL-0005) cells were kindly provided by Procell Life Science & Technology Co., Ltd. Cells were cultured in Dulbecco's modified eagle's medium (DMEM, Hyclone, USA) containing 10% fetal bovine serum (FBS, BI, Israel) (10% DMEM) and maintained in DMEM containing 2% FBS (2% DMEM).
The EMCV NJ08 strain (GenBank: HM641897) was gifted by Professor Jiang Ping of Nanjing Agricultural University. Virus was replicated and harvested using BHK-21 cells infected with EMCV. The titer of 108.5 TCID50/mL was determined by MTT assay according to the method of Reed-Muench .
The recombinant plasmid containing the EMCV 3D gene was generated in the laboratory . Curcumol and ribavirin were purchased from China Food and Drug Control Institute, with 99.9% and 100% purity, respectively. HPCD was purchased from Sigma-Aldrich, with 100% purity. The dissolution of curcumol in DMEM requires 1% DMSO as a co-solvent, while ribavirin and HPCD are directly dissolved in DMEM.
STAT2 Rabbit Polyclonal antibody, JAK1 Ribbit Monoclonal antibody, IRF9 Rabbit Polyclonal antibody, GAPDH Mouse Monoclonal antibody, Beta Actin Recombinant antibody, Goat anti-Mouse and Goat anti-Rabbit secondary antibodies were purchased from Proteintech (USA). P-STAT2 and CH25H Rabbit Monoclonal antibody were purchased from Abcam (USA). PI4KA and OSBP Rabbit Polyclonal antibody were purchased from ABclonal (USA).
Western blot analysis
The different protein levels in the HEK-293T cells infected with EMCV were detected by WB after the treatment with curcumol. Briefly, HEK-293T cells were seeded in 6-well plate and incubated with 100 TCID50/mL EMCV (2 mL/well) for 1.5 h. The supernatant was discarded and three 2 folds serial dilution of curcumol at starting dilution of 25 μg/mL were added and incubated for 24 h. The control group, virus group ribavirin positive control group (0.25 mg/mL) and HPCD (5 mg/mL) positive control group were applied.
The cells were lysed with RIPA buffer containing 1 mM protease inhibitor, 1 mM phosphatase inhibitor and cells were collected using a cell scraper. Total cell protein was extracted and protein concentration was determined using the BCA protein assay kit (Beyotime Biotechnology, Jiangsu, China). Equal amount of cell lysate was separated on a 10% SDS-polyacrylamide gel and transfer to a tailored polyvinylidene fluoride (PVDF) membrane according to the size of the protein. Then the membrane was blocked with Tris-buffered Tween 20 (TBST) with 5% non-fat dry milk at 25°C for 2 h. Next, the membrane was incubated with following primary antibodies overnight at 4°C: GAPDH Mouse Monoclonal antibody (1:50000), STAT2 and IRF9 Rabbit Polyclonal antibody (1:1000), PI4KA and OSBP Rabbit Polyclonal antibody (1:2000), JAK1and P-STAT2 Rabbit Monoclonal antibody (1:2000), CH25H Ribbit Monoclonal antibody (1:500). Then, the membrane was washed with TBST three times, and incubated with the membrane with goat anti-mouse and goat anti-rabbit secondary antibodies (1:20000) at 25°C for 2 h. Finally, the target protein was detected by an enhanced chemiluminescence system (Boster, China). Densitometric values of protein bands were quantified by the Image J.
The HEK-293T cells cultured in 6-well plates were infected with 100 TCID50/mL EMCV, treated with same concentration of curcumol as above and incubated for 24 h. Total RNA was extracted from the cell samples, according to the Trizol protocol (Invitrogen, Carlsbad, CA, USA). The RNA concentration and purity were evaluated by Eppendorf BioPhotometer D30 (Eppendorf, USA). The complementary DNA was synthesized using PrimeScript® RT Master Mix kit with gDNA Eraser (TaKaRa, Dalian, China) according to the manufacturer’s protocol. Q-PCR was performed using a 7500 Real Time PCR System (ABI, USA). Absolute q-PCR was applied to determine EMCV 3D gene level against the standard curve generated using serially diluted plasmid containing 3D gene. The primer sequences used for EMCV 3D gene were: F 5’-TTAGGGCGGGTTTG TAT-3’, R 5’-TTTGTTAGCGGGAGTTA-3’.
The HEK-293T cells infected with 100 TCID50/mL EMCV (2 mL/well) and treated with 0.025 mg/mL curcumol and 5 mg/mL HPCD were incubated for 24 h. The cells were lysed with RIPA buffer containing 1 mM protease inhibitor, 1 mM phosphatase inhibitor. Cell protein concentration was determined by BCA assay (Beyotime Biotechnology, China). Cell lysates were normalized to equal amounts of protein for measurement of free and esterified cholesterol by the Amplex Red kit (Invitrogen, USA). The cholesterol standard was prepared following the manufacture’s instruction with cholesterol concentrations of 0 to 4 μg/mL (0 to 10 μM). 50 μL cell lysates diluted with reaction buffer was added to a microplate, followed by 50 μL working solution of 300 μM Amplex® Red reagent. The microplate was incubated at 37oC for 30 min and protected from light. The fluorescence was measured using a fluorescence microplate reader (Thermo Fisher Scientific, USA) with excitation at 560 nm and emission detection at 590 nm.
Transmission electron microscope
The HEK-293T cells were cultured in 6-well plates, infected with 100 TCID50/mL EMCV (2 mL/well) and allowed to adsorb for 1.5 h. The supernatant was discarded, fresh medium with 0.025 mg/mL curcumol and 5 mg/mL HPCD were added for further incubation in a 5% CO2 37°C cell incubator for 24 h. Meanwhile, control group, virus group were applied and incubated for 24 h. The cells were collected in a 1.5 mL centrifuge tube, and 2.5% glutaraldehyde solution was added (the centrifuge tube was filled with fixative solution so that the sample was completely immersed in the fixative solution), and the cells were fixed for 12 h. Pour off the fixative, rinse the sample three times with 0.1 M, pH 7.0 phosphate buffer for 15 min each time; fix the sample with 1% osmic acid solution for 2 h; carefully remove the osmic acid waste solution, rinse the sample three times with 0.1 M, pH 7.0 phosphate buffer for 15 min each time. The samples were dehydrated using increasing concentrations of ethanol (30%, 50%, 70%, 80%, 90% and 95%) for 15 min with each concentration and then treated with 100% ethanol for 20 min; finally transition to pure acetone treatment for 20 min. The samples were treated with the mixed V/V=1/1 solution of embedding medium and acetone for 1 h, the mixed V/V=3/1 solution for 3 h, and the pure embedding agent for overnight. The infiltrated samples were embedded and heated at 70°C overnight to obtain embedded samples. The samples were sectioned in an ultramicrotome (LEICA EM UC7 type) to obtain 70-90 nm sections, which were stained with lead citrate solution and uranyl acetate 50% ethanol saturated solution for 10 min each. After drying, it can be observed in a transmission electron microscope (Tecnai Spirit Bio-TWIN, FEI, USA).
All data were presented as Mean ± SD. Data were analyzed using GraphPad PrismTM software 5.0 (GraphPad Software, Inc. California, USA). Densitometric values of protein bands were quantified by the Image J. Data were analyzed using GraphPad Prism™ software 5.0 (GraphPad Software, Inc. California, USA). One-way analysis of variance (ANOVA) followed by a Dunnett’s post-test was used to determine the difference between the groups. All groups are compared with EMCV-infected group, * P < 0.05, ** P < 0.01, *** P < 0.001.