Reagents, cell lines, plasmids, and viruses.
PDTC, sodium diethyldithiocarbamate (DDTC), pyrrolidine, N-acetyl cysteine (NAC), apocynin (APO), ATP, ZnCl2, EDTA, EDTA-Ca, EDTA-Mg, EDTA-Zn, EDTA-Cu, N,N,N′,N′-tetrakis (2-pyridylmethyl) ethylenediamine (TPEN), and pyrazone-41 (PYR-41) were purchased from Sigma-Aldrich (St. Louis, MO). WP1130 (Degrasyn) was obtained from Selleckchem (Houston, TX). 2-Dichlorodihydrofluorescein diacetate (DCFH-DA), MG132, SB203580, SP600125, antibodies for IκB-α, anti-p38 antibody, and anti-phosphorylated p38 (p-p38) antibody were from Beyotime Institute of Biotechnology (Haimen, Jiangsu, China). Goat anti-mouse Alexa Fluor 488-IgG, 4′,6′-diamidino-2-phenylindole (DAPI), and FluoZin-3 AM were from Life Technologies (Carlsbad, CA). IRDye 680 and IRDye 800 secondary antibodies were obtained from LI-COR (Lincoln, NE). Antibodies for the late protein gD-1/2 (gD of HSV-1 and -2), ICP0-1, ICP4-1, p53, p65, phosphorylated Jun N-terminal protein kinase 1 and 2 (p-JNK1/2), JNK2, PML, glyceraldehyde-3-phosphate dehydrogenase (GAPDH), β-catenin, and radioimmunoprecipitation assay (RIPA) lysis buffer were purchased from Santa Cruz (Santa Cruz, CA). Antiubiquitin antibody was obtained from eBioscience (San Diego, CA). Anti-HSV VP5 antibody was from Abcam (Cambridge, MA). Bz-VGR-AMC, Suc-LLVY-AMC, and Z-LLE-AMC (where Bz is benzoyl, Suc is N-succinyl, Z is benzyloxycarbonyl, AMC is 7-amido-4-methylcoumarin, and V, G, R, L, V, Y, and E are amino acids) were synthesized by GL Biochem Ltd. (Shanghai, China).
HEC-1-A, HeLa, and Vero cells were obtained from American Type Culture Collection (ATCC, Manassas, VA). Vero-ICP10P, an HSV-2 infection indicator cell line, was generated from Vero cells stably transfected with an HSV-2 ICP10 promoter-driven luciferase reporter plasmid. NF-κB-luc and AP-1-luc reporter plasmids were from Clontech (Palo Alto, CA). pRK-Ub-WT (plasmid number 17608; wild-type [WT] ubiquitin expression plasmid) and pRK-Ub-KO (plasmid number 17603; dominant negative [DN] ubiquitin expression plasmid) were from Addgene (Cambridge, MA). HSV-1(HF) and HSV-2(G) were propagated and titrated on Vero cells as described previously (34
). The HSV-2 virus titration assay could also be performed on Vero-ICP10P cells by measuring luciferase activity. The viral titration was calculated from the standard curve.
Cells were lysed and centrifuged at 12,000 × g for 10 min at 4°C. Total protein in supernatant was quantified by using a bicinchoninic acid (BCA) protein assay kit (Pierce, Rockford, IL). After SDS-PAGE separation, the proteins were transferred onto polyvinylidene difluoride (PVDF) membranes (Millipore, Billerica, MA). The membranes were blocked, incubated in primary antibodies for 2 h at room temperature (RT), washed, and then incubated in IRDye IgG (1:10,000) for 1 h at RT. The bands were visualized with a LI-COR Odyssey infrared imager (LI-COR), and the intensities were analyzed using Odyssey version 3.0 software.
In-Cell Western assay.
Cell monolayers in a 96-well plate were fixed with 4% paraformaldehyde for 20 min at RT and permeabilized by 5 washes in 0.1% Triton X-100 with 5 min for each wash. The wells were blocked for 90 min and then incubated with primary antibodies diluted in blocking buffer (1:200) for 2 h. After washing with phosphate-buffered saline with Tween 20 (PBS-T; pH 7.4) buffer, the cell monolayers were inoculated in IRDye-IgG (1:1,500) for 1 h, rinsed, and scanned in the LI-COR Odyssey infrared imager. The fluorescence intensity of each well was measured by using Odyssey version 3.0 software, and the protein expression level was normalized to that of β-catenin.
HEC-1-A cells cultured on 10-mm glass coverslips were rinsed with PBS and fixed with 4% paraformaldehyde for 15 min at RT before being permeabilized using 0.2% Triton X-100 for 15 min. The cells were blocked for 1 h, and cellular proteins were immunolabeled using the respective primary antibodies followed by Alexa Fluor 488-IgG. Nuclei were visualized by staining with DAPI. Images were acquired using an Olympus FluoView FV10i confocal microscope (Tokyo, Japan).
Cell transfection and luciferase assay.
Cells cultured in a 96-well plate were transiently transfected with 100 ng/well luciferase reporter plasmid or cotransfected with 50 ng/well luciferase reporter plasmid and 150 ng/well ubiquitin expression plasmids using Lipofectamine 2000 transfection reagent (Life Technologies). Cells were cultured for a further 24 h and then infected with virus in the absence or presence of drugs. The relative luminescence units (RLU) were determined by using the Bright-Glo luciferase assay system (Promega, Madison, WI).
RNA extraction and real-time PCR.
Total RNA was extracted using TRIzol reagent (Life Technologies) and reverse transcribed to cDNA using the ReverTra Ace quantitative reverse transcription-PCR kit (Toyobo, Osaka, Japan). Real-time PCR was performed in triplicate on the ABI Prism 7300 sequence detection system using SYBR green PCR master mix (Toyobo) according to the manufacturer's protocol. The levels of HSV-1 ICP0 (forward, TACGTGAACAAGACTATCACGGG, and reverse, TCCATGTCCAGGATGGGC), HSV-1 ICP4 (forward, GGCCTGCTTCCGGATCTC, and reverse, GGTGATGAAGGAGCTGCTGTT), HSV-1 gD (forward, AGCAGGGGTTAGGGAGTTG, and reverse, CCATCTTGAGAGAGGCATC), HSV-2 ICP0 (forward, GTGCATGAAGACCTGGATTCC, and reverse, GGTCACGCCCACTATCAGGTA), HSV-2 ICP4 (forward, GCGAGCTGCGGTTCGT, and reverse, GCCACGCGCAGGTC), and HSV-2 gD (forward, CCAAATACGCCTTAGCAGACC, and reverse, CACAGTGATCGGGATGCTGG) mRNA transcription were standardized against that of the GAPDH housekeeping gene (forward, TGCACCACCAACTGCTTAGC, and reverse, GGCATGGACTGTGGTCATGAG).
Cells cultured in black opaque 96-well plates were treated with the antioxidants for 30 min prior to HSV-2 infection (multiplicity of infection [MOI] of 1) for 24 h. The cells were washed with PBS and then exposed to DCFH-DA diluted in Dulbecco's modified Eagle's medium (DMEM) medium (10 μM). After 20 min of incubation, the cells were rinsed 3 times, and the plates were read using the Hitachi F7000 spectrofluorometer (Tokyo, Japan) (excitation, 488 nm, and emission, 525 nm).
Cellular 26S proteasome activity assay.
HEC-1-A cells were lysed, and cytoplasmic extracts were obtained by centrifugation at 12,000 × g
for 10 min at 4°C. The effects of drugs on cellular 26S proteasome activity were determined as described previously (35
). Briefly, 20 μg total protein diluted in assay buffer (20 mM Tris, pH 8.0, 1 mM ATP, and 2 mM MgCl2
) was mixed with the compounds indicated below and with 75 μM substrates to a final volume of 100 μl. The substrates, Bz-VGR-AMC, Suc-LLVY-AMC, and Z-LLE-AMC, were used to determine the proteasome trypsinlike, chymotrypsinlike, and peptidylglutamyl-peptide hydrolase (PDGH) activities, respectively. The mixtures were incubated in the absence or presence of drugs for 1 h at 30°C. 26S proteasome cleavage activity was measured in the Hitachi F7000 spectrofluorometer (excitation, 380 nm, and emission, 460 nm).
Intracellular Zn2+ detection.
HEC-1-A cells grown in black opaque 96-well plates were loaded with 5 μM FluoZin-3 AM in PBS for 30 min at 37°C. The cells were then washed with Zn2+-free medium to remove nonspecific dye staining on the cell surface and subsequently incubated for 30 min in complete medium to allow deesterification. PDTC was added to the medium for 30 min, and the plate was read in the spectrofluorometer (excitation, 494 nm, and emission, 516 nm).
Statistical analysis was performed using the two-tailed Student t test. Statistical significance was determined at P values of <0.05 and <0.01.