Nuclear extracts from Jurkat cells were used as negative control, and nuclear extracts from
Raji cells included in the kit and from MoT cells served as positive controls in the assay. In order to address the potential cytotoxic effects of https://www.selleckchem.com/products/i-bet-762.html pyrrolidine dithiocarbamate (PDTC) on mononuclear cells, experiments were performed treating PBMCs with PDTC for 1 h at three different concentrations (1 μM, 10 μM, 30 μM) or left them untreated, then washed three times with RPMIc. After 3 h in culture, cell viability was measured by the trypan blue exclusion method. PDTC-treated cells were also subjected to apoptosis determination by fluorescence activated cell sorter (FACS) using the annexin-V/7-aminoactinomycin D (7AAD) kit (BD Bioscience Pharmingen). More than 95% viable cells were determined in trypan blue exclusion assay for PBMCs treated with PDTC under these concentrations. In addition, the PDTC agent did not affect the viability of the cells as assessed by annexin-V and 7AAD staining (data not shown), and therefore pretreatment of PBMCs was performed with 30 μM of PDTC. To examine the role of NF-κB in Tax-mediated CC-chemokine secretion, PBMCs were pretreated with 30 μM of PDTC, a potent inhibitor of NF-κB, for 1 h then washed three times with RPMIc, followed by
treatment with Tax proteins (100 pM) for 3 h, shown to be the optimal time-point to assess levels of CC-chemokines in Tax-treated PBMCs (Fig. 1). In other experiments, Pirfenidone manufacturer PBMCs were transduced with the NF-κB super-repressor (NF-κB/SR) at an MOI of 25 using lipofectamine plus reagent (Invitrogen) for 20 h prior to Tax protein treatment (3 h). PBMCs were also co-transduced with NF-κB/SR and Ad-Tax2 or Ad-GFP. Cell-free supernatants were harvested after 24 h of incubation and assayed for MIP-1α, MIP-1β and RANTES expression, as described above. All statistical analyses were performed using GraphPad Prism version 6·00 for Windows (GraphPad
Nitroxoline Software http://www.graphpad.com) and the data expressed as mean ± standard error of the mean. One-way analysis of variance (anova) with Bonferroni’s multiple post-test comparison were used to evaluate three or more groups. Statistical comparisons for two groups were assessed by two samples assuming equal variances Student’s t-test. P-values <0·05 were considered statistically significant. We have reported recently that extracellular Tax2 and Tax1 proteins induced high levels of CC-chemokines in mononuclear cells [24, 25]. The optimal dose of protein required to detect CC-chemokine secretion was determined previously by exposing PBMCs to increased concentrations of Tax proteins ; the concentration of 100 pM was optimal, and therefore used in all subsequent experiments. In order to determine the time of MIP-1α, MIP-1β and RANTES release, PBMCs were treated once with Tax2A (subtype A), Tax1 or mock-treated control and then cell-free supernatants were harvested after 1, 2, 3, 6, 12 or 24 h of incubation.