Reagents, cell lines, plasmids and viruses
Wogonin and acyclovir were obtained from the National Institutes for Food and Drug Control in China (Beijing, China). SB203580, SP600125, and MG132 were purchased from Beyotime Biotechnology Institute (Haimen, Jiangsu, China). Alexa Fluor 488-conjugated goat anti-mouse IgG (H + L), DAPI, DRAQ5 and SYBR green real-time PCR reagent were obtained from Life Technologies, Thermo Fisher Scientific (Carlsbad, CA, USA). IRDye 680-conjugated goat-anti-rabbit and IRDye 800-conjugated goat-anti-mouse antibodies were obtained from LI-COR (Lincoln, NE, USA). Antibodies specific for HSV-1/2 gD, HSV-1 ICP0, HSV-1 ICP4, HSV-1 ICP27, JNK2, p38, GAPDH, and RIPA lysis buffer were purchased from Santa Cruz (Santa Cruz, CA, USA). p65, p-p38, p-c-Jun, p-JNK1/2, p-ATF-2, and IκB-α antibodies were purchased from Cell Signaling Technology (Beverly, MA, USA). Bright-Glo luciferase assay system was purchased from Promega (Madison, WI, USA).
Vero and HEC-1-A cells were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA). NF-κB-luc and AP-1-luc reporter plasmids were purchased from Clontech (Palo Alto, CA, USA). HSV-1(HF), HSV-1/blue and HSV-2 (G) were propagated and titrated in Vero cells as described previously [36].
In vitro viral inhibition assay
The in vitro viral inhibitory effects of wogonin were determined via titration of infectious virions according to a previously described method [37]. Briefly, confluent HEC-1-A cells in 96-well plates were pretreated with serial concentrations of wogonin for 30 min and then infected with HSV-1 or HSV-2 (MOI = 1). At 24 h p.i., the culture medium on treated HEC-1-A cells was replaced with fresh medium. The infected cells were frozen and thawed in 3 cycles to release the virions. The virions-containing medium was dispensed on confluent Vero monolayer cells, and the viral titration was performed by counting the numbers of plaques after 48 h.
In vitro cytotoxicity assay
In vitro cytotoxicity was determined using a commercial Cell Counting Kit-8 (CCK-8, Dojindo, Kumamoto, Japan) according to the manufacturer’s instructions. Briefly, cells were seeded at a density of 2 × 104 cells per well into 96-well plates, cultured for 24 h, and then treated with serial dilutions of compound in triplicate. After 24 h of incubation, 10 μl of CCK-8 reagent was added to each well, and the plates were incubated at 37 °C for 3 h. The absorbance at 450 nm was measured using a Tecan Infinite M200 microplate reader (Männedorf, Switzerland), and cell viability was plotted as the percentage of viable cells compared with that of the mock-treated control cells.
Western blot and in-cell western assays
Western blot and in-cell western assays were performed as described previously [20]. Cells were lysed using RIPA lysis buffer on ice for 30 min and then centrifuged at 12,000×g for 10 min at 4 °C to collect the supernatants. The total protein concentrations were determined using a BCA protein assay kit (Thermo Fisher Scientific, Waltham, MA, USA). After separation via SDS-PAGE, the proteins were electrotransferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA, USA), The membrane were blocked using Odyssey blocking buffer (LI-COR) and primary and incubated with secondary antibodies, and the protein bands were visualized via an Odyssey Infrared Imager (LI-COR).
An in-cell western assay was performed in a 96-well plate. Cells were fixed with 4% paraformaldehyde for 20 min at room temperature (RT) and permeabilized via five washes in 0.1% Triton X-100 in phosphate-buffered saline (PBS) for 5 min per wash. The cell monolayers were blocked for 90 min in blocking buffer consisting of4% nonfat dry milk in PBS with 0.1% Tween-20 (PBS-T) and then incubated with primary antibodies diluted in blocking buffer (1:200) for 2 h at RT. After washing with PBS-T, the cell layers were stained with IRDye IgG (1:1500) for 1 h. The plate was rinsed and scanned in an Odyssey Infrared Imager. The relative protein expression levels were normalized to those of DRAQ5.
Time-of-drug-addition assay
A time-of-drug-addition assay was carried out via measurement of HSV-2 gD protein expression levels, which can indicate viral replication efficiency. HEC-1-A cells were seeded into a 96-well plate and infected with HSV-2 (MOI = 1). Wogonin or other drugs with known inhibitory mechanisms were dispensed at different time points. The viral gD levels were determined via in-cell western assay at 24 h p.i. as described previously [38].
Cell transfection and luciferase assays
HEC-1-A cells were transiently transfected with luciferase reporter plasmids using Lipofectamine 2000 transfection reagent (Life Technologies, Thermo Fisher Scientific). The relative luminescence units (RLUs) were determined using a Bright-Glo luciferase assay system (Promega). Briefly, HEC-1-A cells were seeded into 96-well plates. When the confluence reached ~ 90%, the cells were transfected with 100 ng of NF-κB or AP-1 luciferase reporter plasmid. The cells were subsequently cultured for 24 h and then treated with inhibitors for another 24 h. The luminescence signals were monitored with GloMax-96 microplate luminometer (Promega).
RNA extraction and quantitative PCR
Total RNA was extracted using TRIzol reagent (Life Technologies, Thermo Fisher Scientific) according to the manufacturer’s instructions. Complementary DNA (cDNA) was reverse-transcribed using a ReverTra Ace qPCR RT kit (Toyobo, Osaka, Japan). Real-time qPCR was performed in triplicate on an ABI Prism 7300 Sequence Detection System using SYBR Green PCR Master Mix (Life Technologies). The sequences of the primers used in this study are as follows: HSV-1 gD, 5′-AGCAGGGGTTAGGGAGTTG-3′ (Forward) and 5′-CCATCTTGAGAGAGGCATC-3′ (reverse); HSV-2 gD, 5′- CCAAATACGCCTTAGCAGACC-3′ (forward) and 5′-CACAGTGATCGGGATGCTGG-3′ (reverse); human GAPDH, 5′-TGCACCACCAACTGCTTAGC-3′ (Forward) and 5′- GGCATGGACTGTGGTCATGAG-3′ (reverse). The mRNA transcription levels were standardized against those of the housekeeping gene GAPDH.
HSV-1/blue assay
An HSV-1/blue assay was performed as previously described with modifications [39]. The confluent HEC-1-A cells in a 96-well plate were preincubated with serial dilution of drugs for 30 min at 37 °C. The cells were then infected with HSV-1/blue (MOI = 1). The cells were lysed with 1% NP-40 in DMEM 12 h p.i. Cell lysates from each well were then transferred into a new Costar 96-well flat plate. The β-gal substrate solution chlorophenol red-β-D-galactopyranoside (CPRG) was added to each well. The absorbance at 570 nm was measured after 1 h using a Tecan Infinite M200 microplate reader.
Immunofluorescence staining and confocal microscopy
HEC-1-A cells were seeded onto 10 mm glass coverslips, which were then placed in a 24-well plate. The cells growing on the coverslips were rinsed with PBS, fixed with 4% paraformaldehyde for 15 min at RT, and permeabilized with 0.2% Triton X-100 for 15 min. The coverslips were blocked with 1% BSA in PBS for 30 min at RT. Target biomarkers were immunolabeled using appropriate primary antibodies and Alexa Fluor 488-conjugated secondary antibody. Nuclei were visualized by staining with DAPI. Images were acquired using an Olympus FluoView FV10i confocal microscope (Tokyo, Japan).
Drug synergism analysis
We employed an in-cell western assay to determine the efficiency of wogonin- and acyclovir-mediated inhibition of HSV-2 gD expression in HEC-1-A cells. The half maximal effective concentration (EC50) of the two compounds were calculated. Combinations of the compounds with fixed molar ratios were also investigated. The ratio was optimized to yield the greatest synergism over a range of serial dilutions. The combination index (CI) of the two drugs was calculated in CalcuSyn software (Biosoft, Cambridge, UK) with a method described by Chou and Talalay [40]. Synergy was assessed according to the CI values and scored as follows: CI < 0.1, very strong synergism; CI = 0.1 to 0.3, strong synergism; CI = 0.3 to 0.7, synergism; CI = 0.7 to 0.85, moderate synergism; CI = 0.85 to 0.90, slight synergism; CI = 0.9 to 1.1, nearly additive synergism; and CI = > 1.1, antagonism.