Figure 5From: Reduced levels of reactive oxygen species correlate with inhibition of apoptosis, rise in thioredoxin expression and increased bovine leukemia virus proviral loadsEffect of VPA on apoptosis and ROS production. PBMCs from BLV-infected and control sheep were isolated and cultivated for 24 h in the absence (0) or the presence of 1 mM of VPA. A) Representative Western blot analysis using an antibody specific for the acetylated form of histone H3. Actin was analyzed in parallel as a loading control. B) The extent of B cell apoptosis was measured by determining the level of nuclear DNA fragmentation. B lymphocytes from 6 BLV-infected and control sheep were labeled using an anti-IgM monoclonal antibody (clone 1H4) and a FITC-conjugated rabbit anti-mouse antiserum (Becton Dickinson). After ethanol fixation and propidium iodide staining, hypodiploid B lymphocytes (i.e. in sub-G1) considered to be apoptotic were quantified by flow cytometry. ** denotes the statistical significance of the differences between 0 and 1 mM VPA, accordingly to the paired Student's t test p < 0.01. C) ROS levels were evaluated in parallel after incubation of PBMCs with 10 μM of CM-H2DCFDA probe prior to VPA treatment. Data represent the mean fluorescence intensities (± standard deviation) of cellular chloromethyldichlorofluorescein (CM-DCF). * and ** denote the statistical significance of the differences between 0 and 1 mM VPA, accordingly to the paired Student's t test (p < 0.05 and p < 0.01, respectively). D and E) PBMCs from BLV-infected and control sheep were cultivated for 24 h in absence or presence of the free radical scavenger N-acetyl-L-cysteine (NAC) added 2 h prior VPA treatment (1 mM). Cells isolated from BLV-infected and control sheep were analyzed by flow cytometry to determine the levels of intracellular ROS (D) and the rates of apoptosis (E). ** and *** denote the statistical significances according to the paired Student's t test (p < 0.01 and p < 0.001, respectively).Back to article page