Lenalidomide actin mRNA in each individual sample was used to normalize the dataset

Two EBV B cell lines, BJAB and Toledo, originally generated Taxifolin from a BL30 and a non Hodgkin lymphoma,31 respectively, were also used in this study. Except for BJAB, all cells were maintained in RPMI 1640 with 10% FBS containing 100 U of penicillin and 100 g of streptomycin per milliliter. BJAB cells were maintained in DMEM with 20% FBS Cidofovir molecular weight and antibiotics. HDAC inhibitors A complete list of HDAC inhibitors used in this study is provided in Table 1 and their structures are shown in Figure 1. Sodium butyrate and SAHA were purchased from Sigma Aldrich. Valproic acid, Scriptaid, apicidin, and oxamflatin were purchased from Calbiochem. MS275, LBH589, PXD101, and various largazole derivatives32 were synthesized at the Department of Chemistry, Colorado State University.
Stock solutions of sodium butyrate were prepared fresh in sterile water. Valproic acid stock was prepared in water with the pH adjusted to 7.5 with 1M NaOH. Stock solutions of all other HDAC inhibitors were prepared in DMSO and stored in aliquots at 20°C. Analysis of lytic gene expression To determine EBV lytic phase gene expression, Marbofloxacin price levels of thymidine kinase and BGLF4 gene transcripts were analyzed by reverse transcription and real time PCR. Total RNA from P3HR1, Daudi, or JY cells, either untreated or treated with various HDAC inhibitors, was isolated by TRIzol reagent extraction following the manufacturer’s protocol. Genomic or episomal DNAcontamination from the RNApreparations were removed by RNase free DNase treatment at a concentration of 0.1 U/L.
Five micrograms of total RNA was reverse transcribed by Superscript III using random hexamer primers. The TK, BGLF4, or actin cDNA from these preparations was then amplified using SYBR Green PCR amplification technology in an ABI PRISM 7500 sequence sodium butyrate ic50 detection system . The primers used in the real time PCR amplification are listed in Table 2. Relative quantification of gene expression was determined by the comparative threshold method , as described previously.33 Expression of the actin mRNA in each individual sample was used to normalize the dataset. PCR assay for the presence of EBV The presence of EBV genome in the cell lines used in the study was determined by PCR analysis of EBV EBER1 sequences in the genomic and To investigate the efficiency of the several newer HDAC inhibitors for the induction of EBV lytic phase gene expression, we measured their effect on the expression of the gene encoding the viral TK enzyme.
The EBV TK enzyme is normally expressed only during the lytic replication stage of the virus life cycle. We initially studied the EBV BL cell line P3HR1, which typically maintains a type I latency in which only the EBV latency genes nurse EBNA1, EBER1, and EBER2 are expressed. The cells were exposed to individual HDAC inhibitors for 48 hours, followed by total RNA extraction. TK transcript expression in these preparations was then analyzed by reverse transcription and real time PCR analysis. Exposure to most of the HDAC inhibitors produced increases in TK expression in a dose dependent manner compared with the level of expression in vehicle treated control cells . The highest level of increase in TK expression was produced by the hydroxamic acids LBH589 and PXD101 .