Figure 5
Effect 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).