The STAT3 inhibitor WP1066 and the JAK inhibitor pyridone six have been purchased from Merck. Neutralising anti EGFR and anti IL 6R antibodies had been obtained from Millipore and R&D Systems respectively. Recombinant glycoprotein gB was purchased from Abcam. Ganciclovir was bought from Roche. Cell culture HepG2 cells were obtained from the European Collection of Cell Cultures and PHH from Kaly Cell. HepG2 cells have been cultivated in Eagles Minimum Essential Medium supplemented with 10% fetal bovine serum, 1% non essential amino acids, penicillin, and streptomycin. PHH were cultivated in serum free Dulbeccos Modified Eagle Medium supplemented with L glutamine, insulin, dexamethasone, and gentamycin. The PHH were free of HCV, HBV, HIV, and HCMV as determined by highly sensitive PCR and RT PCR assays. Cell viability assay was performed as previously described. IL 6 production was measured in culture supernatants using an ELISA kit.
Quantification of HCMV titers in cell culture supernatants was performed by real time PCR as previously described. HCMV infection of HepG2 selleck chir99021 cells and primary human hepatocytes Cell free virus stock was prepared by propagating two strains of HCMV, the laboratory strain AD169 and a clinical isolate, HCMV DB, in MRC5 human fibroblasts as described previously. AD169 is a highly passaged laboratory strain of HCMV originally isolated from the adenoids of a child. The clinical isolate HCMV DB was isolated from a cervical swab specimen from a 30 year old pregnant woman. MRC5 human fibroblasts have been cultured in EMEM with 10% FBS, penicillin, and streptomycin. HepG2 cells and PHH have been infected at different multiplicities of infection for 2 h at 37uC, washed thoroughly, and covered with fresh medium.
Where specified, cells were treated with ganciclovir during infection with HCMV. Ultraviolet inactivated HCMV was used as control. Supernatants were clarified by centrifugation and stored at experienced 280uC until use. Virus titers had been determined by plaque forming assay in MRC5 human fibroblasts as described previously. RT PCR assay Briefly, total RNA was extracted from HepG2 cells with RNeasy mini kit. RNA was reverse transcribed into cDNA with Superscript III RT using oligo primers. The RT product was used to perform PCR of IE 1 exon 4 and US28 transcripts with following pairs of primers. The PCR product was analysed on a 2% agarose gel and visualized after staining with ethidium bromide. For quantitative RT PCR, 2 microl cDNA product was used in 50 microl cDNA amplification reaction with 300 nM of IE 1 and US 28 primers together with syber green PCR master mix.
The reaction had been set up in MicroAmp optical 96 effectively reaction plate, sealed and cycled on Stratagene MX3005P realtime qPCR system with 95uC for 10 min, followed by 40 cycles at 95uC for 15 sec and annealing/extension on 60uC for 1 min.