2.1. Cell culture and virus infection
BCBL-1 cells (KSHV-positive and EBV-negative PEL cell lines) were obtained through acquired immunodeficiency syndrome (AIDS) Research and Reference Reagent Program, National Institutes of Health. Vero cells (African green monkey kidney fibroblasts) were obtained from American Type Culture Collection (ATCC). BCBL-1 and Vero cells were maintained in RPMI-1640 and Dulbecco's modified Eagle's medium (DMEM) respectively, both of which contained 10% fetal bovine serum (FBS), 2 mmol/l L-glutamine, 100 U/ml penicillin, and 100 μg/ml streptomycin at 37°C in a humidified, 5% CO2 atmosphere. HSV-1 (McKrae strain) was propagated and viral titers were determined in Vero cells as described previously . The supernatant from normal Vero cells culture was used as a control (Mock). Before infection or transfection, BCBL-1 cells were incubated in serum-free RPMI-1640 medium for a maximum inducibility of KSHV replication .
2.2. Antibodies and reagents
Anti-phospho-STAT3 (Tyr705) rabbit monoclonal antibody (mAb), anti-phospho-PI3K p85 (Tyr458)/p55 (Tyr199) rabbit polyclonal antibody (pAb), anti-phospho-AKT (Ser473) mouse mAb, anti-phospho-GSK-3β (Ser9, GSK: glycogen synthase kinase) rabbit pAb, anti-phospho-c-Raf (Ser338) rabbit pAb, anti-phospho-MEK1/2 (Ser217/221, MEK: MAPK-ERK kinase) rabbit pAb, anti-phospho-ERK1/2 (Thr202/Tyr204) rabbit mAb, anti-STAT3 rabbit pAb, anti-PI3K p85 rabbit pAb, anti-GSK-3β rabbit mAb, anti-c-Raf rabbit pAb, anti-MEK1/2 rabbit pAb, anti-Flag M2 mouse mAb, anti-hemagglutinin (HA) rabbit mAb and LY294002 (PI3K inhibitor) were purchased from Cell Signaling Technologies (Beverly, MA, USA). Anti-PTEN (PTEN: phosphatase and tensin homologue deleted on chromosome ten) mouse mAb, anti-β-actin mouse mAb, anti-α-Tubulin mouse mAb, anti-GAPDH mouse mAb and horseradish peroxidase (HRP)-conjugated goat anti-mouse/rabbit IgG were obtained from Santa Cruz Biotechnologies (Santa Cruz, CA, USA). Anti-AKT rabbit pAb were obtained from BioVision (Mountain view, CA, USA). Anti-ERK1/2 rabbit pAb were obtained from Shanghai Kangchen Biotechnologies (Shanghai, China). Piceatannol (JAK1 inhibitor) was purchased from BIOMOL Research Laboratories Inc. (Plymouth Meeting, PA, USA). Both anti-phospho-STAT6 (Tyr641) mouse mAb and Peptide II (ERK inhibitor) were obtained from Calbiochem (Darmstadt, Germany). Anti-STAT6 rabbit pAb was purchased from Bethyl Laboratories Inc. (Montgomery, TX, USA). Anti-KSHV ORF59 mAb and viral IL-6 (vIL-6) rabbit pAb were obtained from Advanced Biotechnologies Inc. (Columbia, MD, USA). Anti-KSHV Rta (replication and transcription activator) antibody was generated by immunization of rabbits with ORF50 peptide (amino acids 667-691) .
2.3. Western blot analysis
After infection, cells were harvested and lysed in RIPA buffer containing protease and phosphatase inhibitors. 60-80 μg of proteins were loaded onto sodium dodecyl sulphate-polyacrylamide gel electrophoresis (SDS-PAGE), transferred to polyvinylidene fluoride (PVDF) membrane. The membrane was incubated with diluted primary Abs for overnight at 4°C, and then incubated with HRP-conjugated species-specific second Abs for 1 h at 37°C. Proteins were visualized by enhanced chemiluminescence (ECL) reagents (Cell Signaling Technologies) according to the manufacture's instructions.
2.4. RNA isolation and real-time quantitative PCR (RT-qPCR)
Total RNA was isolated from cells by using Trizol reagent (Invitrogen, Carlsbad, CA). RT-qPCR was performed in a GeneAmp 7300 sequence detection machine (Applied Biosystems, Foster City, CA) as described previously . The sequences of KSHV ORF26 primer and probe were listed as described previously .
2.5. Plasmids and transfection
The dominant negative STAT3 construct (pMSCV-STAT3 dominant negative-GFP, abbreviated pST3-DN) was kindly provided by D. Link (Washington University School of Medicine, MO, USA) . The dominant negative STAT6 construct (pDsRed1-N1-STAT6 dominant negative-RFP, abbreviated pST6-DN), containing amino acids 1-661 of STAT6, was a kind gift of K. Zhang (UCLA School of Medicine, CA, USA) . The dominant negative construct of PI3K (P85σiSH2-N, designated as PI3K-DN in this study), the dominant negative construct of AKT (SRα-AKT, designated as AKT-DN), and corresponding control vectors pSG5 and pSRα were generously provided by B-H Jiang (Nanjing Medical University, Nanjing, China) . The dominant negative MEK1/2 construct (MEK-DN) was presented as a gift by G. Chen (Medical College of Wisconsin, WI, USA). The protein expressing plasmid of GSK-3β (GSK-3β-S9A, there was a tag of HA) was purchased from Addgene (http://www.addgene.org). The PTEN cDNA plasmid (there was a tag of Flag) was constructed in our lab. BCBL-1 cells were electroporated at 250 V and 960 μF using a Gene Pulser (Bio-Rad Laboratories, Hercules, CA) as described elsewhere .
2.6. Detection of the release of KSHV progeny virions
After BCBL-1 cells were infected with HSV-1 for 48 h, supernatant from cell cultures was harvested and filtered through a 0.45-μm-pore-size filter. The filtered supernatant was centrifugated for 30 min at a speed of 15 000 rpm at 4°C and the precipitation contained KSHV progeny virions. The virions were resuspended in PBS and viral DNA was extracted using the high pure viral nucleic acid kit (Roche, Germany) as per the manufacturer's instructions. Purified viral DNA was used for real-time DNA-PCR analysis. The KSHV ORF26 gene cloned in the pcDNA3.1 (abbreviated pcDNA, Invitrogen) was used to generate the standard curve.
2.7. Immunofluorescence assay (IFA)
IFA was performed as described elsewhere . Briefly, after HSV-1 infection, BCBL-1 cells were washed and smeared on chamber slides. Slides were incubated with a 1:100 dilution of anti-KSHV ORF59 mouse mAb. Alexa Fluor 568 (Invitrogen)-conjugated goat anti-mouse antibody (1:200 dilution) was used as a secondary antibody for detection. The cells were counterstained with 4','-diamidino-2-phenylindole. Images were observed and recorded with a Zeiss Axiovert 200 M epifluorescence microscope (Carl Zeiss, Inc.). Photographs of at least 10 unique fields were taken of every slide, and the number of positive and negative cells was counted separately by three individuals, including one who was blinded to the results to calculate the percentage of positive cells.