Reagents and kits
Dulbecco’s Modified Eagle’s Medium (DMEM), fetal bovine serum (FBS), SYBR Green, penicillin, streptomycin, L-glutamine, trypsase, dimethlsulfoxide (DMSO), Triton X-100, phenylmethanesulfonyl fluoride (PMSF), â-mercaptoethanol (â-ME), TNF-α detection kit, IL-6 ELISA kit, and cell cycle assay kit were obtained from Sigma-Aldrich (St. Louis, MO, USA). Mouse anti-HCMV IE and mouse anti-HCMV UL83 were purchased from Abcam, USA. Secondary antibody goat anti-mouse and Platinum® Quantitative PCR SuperMix-UDG were obtained from Invitrogen, USA. RNeasy Mini Kit and Sensiscript Reverse Transcriptase Kit were purchased from Qiagen (Valencia, CA, USA).
Cell culture and virus propagation
Human embryonic lung fibroblast (HELF) cells were purchased from National Platform of Experimental Cell Resources for Sci-Tech (Beijing, China) and cultured in DMEM supplemented with 10% FBS, 100 U of penicillin/ml, 100 ìg/ml of streptomycin, and 2 mML-glutamine at 37°C under a humidified condition of 95% air and 5% CO2. HCMV laboratory strain AD169 was purchased from ATCC, prepared in HELF cells and maintained in DMEM with 2% FBS (maintenance medium). The maximal atoxic concentration (TC0) and half maximal inhibitory concentration (IC50) of curcumin cytotoxicity and GCV and viral titer were determined by plaque titration as described previously [13, 14].
Antiviral compounds
Curcumin was obtained from National Institute for Food and Drug Control (Beijing, China) and stored as 50 mM stock solution in DMSO for in vitro use. The high dose, middle dose, and low dose of curcumin were 0.8, 0.4, and 0.2 ìg/ml, respectively. The intravenous formulation of GCV (Sigma-Aldrich, St. Louis, MO, USA) at 50 ìg/ml in 0.9% saline was used as reference.
Enzyme-linked immunoabsorbent assay (ELISA)
HELF cells that were seeded at density of 1 × 106/ml in tissue culture flasks were infected with HCMV at 100TCID50/0.1 ml. After incubation at 37°C for 2 h under a humidified condition of 95% air and 5% CO2, the supernatant was removed and replaced by the maintenance medium with or without curicumin. After 48 hours, the supernatants were harvested and the levels of inflammatory cytokines IL-6 and TNF-α were detected.
Flow cytometry
HELF cells that were seeded at density 1 × 106/ml in tissue culture flasks were synchronously cultivated for 12 h, and then infected with HCMV at 100TCID50/0.1 ml. After incubation at 37°C for 2 h under a humidified condition of 95% air and 5% CO2, the supernatant was removed and replaced by the maintenance medium with or without curcumin. After 48 h, the cells were harvested, centrifuged at 800 g for 5 min, washed with cold PBS two times, centrifuged at 800 g for 5 min, and re-suspended with 70% ethanol at 4°C overnight. The cells were centrifuged at 800 g for 5 min, washed with PBS three times and incubated with 500 ìl of PBS containing 50 ìg/ml propidium iodide (PI), 100 ìg/ml RNase A and 0.2% Triton X-100 at 4°Cin a dark cabinetfor 30 min.
SYBR green reverse transcriptase real-time quantitative PCR
Total RNAs were extracted from cells and treated with Dnaseusing a QiagenRNeasy kit according to the manufacturer’s instructions. RNA (500 ng) was subjected to reverse transcription. Each reaction mixture contained a cDNA template, 10 μM of forward primer and reverse primer [17, 18] (Oligonucleotide primers (Beijing DingguoChangsheng Biotechnology CO., Ltd, Beijing, China) are as follows: IE forward 5′-AGACACCCGTGACCAAG-3′, IE reverse 5′-TCTGTTTGACGAGTTCTGC-3′; UL83 forward 5′-ATGGTGGCTACGGTTCA -3′, UL83 reverse 5′-CCTCGGTGCTTTTTGG-3′; GAPDH forward 5′-AGACACCCGT GACCAAG-3′, GAPDH reverse 5′-TTTGAGGGTGCAGCGAACTT-3′), 2 × Mix, 10 × Sybr Green I and dH2O. Amplification was performed by a cycle of initial denaturation at 94°C for 2 min; 40 cycle of denaturation at 94°C for 30 s, annealing at 61°C for 30 s, and elongation at 72°C for 30 s. The fluorescence threshold value was calculated using ABI 7700 device system software (Applied Biosystems Inc., Foster City, CA, USA). The calculation of relative change in mRNA was performed using the delta-delta method [19], with normalization to GAPDH.
Immunofluorescence assay
Cells that were seeded on non-coated glass coverslips at a density of 1 × 106/ml were washed with PBS three times and fixed with 4% paraformaldehyde at room temperature (RT) for 15 min. Cells were washed with PBS three times, each time 5 min, followed by permeablization in 0.5% Triton X-100/PBS for 8 min. The cells were washed with PBS and blocked in blocking buffer (10% goat serum in PBS) at 37°C for 1 h. After blocking, the cells were incubated with primary mouse monoclonal anti-human IE and UL83 antibodies in a moist chamber at 37°C for 1 h. Then the cells were rinsed with PBS and incubated with secondary goat anti-mouse fluorescein-conjugated antibody at 37°C for 1 h in a dark cabinet, followed by successive washes with PBS and imaged at 200× using an Olympus microscope (Olympus IX71, Japan). The acquired images were processed using Adobe Photoshop software.
Western blotting
The cells were washed with cold PBS twice and lysed in an appropriate volume of cold RIPA buffer [25 mM Tris–HCl pH 7.6, 150 mM NaCL, 1% NP-40, 1% sodium deoxycholate, 1 mM PMSF and 0.1% sodium-dodecyl sulphate (SDS)] on ice for 30 min; then they were crushed at 4°C and centrifuged at 9600 g for 10 min. The supernatant was removed and denatured by heating at 95°C for 10 min. Proteins were separated by SDS-PAGE (Mini-Protean®Tetra System, Bio-RAD, Hercules, CA, USA), electroblotted onto nitrocellulose membranes (Mini-Trans-Blot, Bio-RAD, Hercules, CA, USA). After being blocked with 5% skim milk in Tris-buffered saline (TBS) consisting of 100 mmol/L Tris · HCL, 150 mmol/L NaCL, pH 7.4 at room temperature for 2 h, the blots were incubated with primary antibodies at the recommended dilutions at 4°C overnight, followed by washing with 0.05% Tween-20 in TBS (TBST) three times. Then the blots were then incubated with Alexa Fluor®680 goat anti-mouse IgG and goat anti-rabbit IgG at 1:10000 (Invitrogen, Grand Island, NY, USA) in TBST. After washing the membranes with TBS, signals were detected using Odyssey infrared laser imaging system (Li-COR, American) according to the manufacturer’s instructions. For densitometric analysis of western blot images, density profiles of the bands were measured using ImageJ software.
Statistical analysis
Data were analyzed using Microsoft Office Excel 2003 and expressed as mean ± standard error (SE). Group comparisons were evaluated using the one-way ANOVA, and significant differences between treatments were determined using the post hoc test with Tukey-Kramer HSD simultaneous pair wise main comparison. P values less than 0.05 were considered to be statistically significant.