Journal: Redox Biology
Article Title: HIV-1 Tat protein induces DNA damage in human peripheral blood B-lymphocytes via mitochondrial ROS production
doi: 10.1016/j.redox.2017.11.024
Figure Lengend Snippet: Tat activates NF-κB in B-cells. (A) Tat induces nuclear translocation of NF-κB in B-cells. The Y axis shows the number of cells exhibiting NF-κB/p65 nuclear staining in B-cells treated with 250 ng/ml of Tat for 6 h, compared to the untreated control, for which the obtained basal value was set as 1. A minimum of 100 cells were analyzed per condition. The experiment was carried out on blood from one healthy donor. (B) Tat-induced nuclear translocation of phosphorylated NF-κB RelA/p65 subunit in normal B-cells. Right panel: Western blot analysis of cytosolic and nuclear fractions from normal B-cells incubated or not with 250 ng/ml Tat for 6 h. α-tubulin was used as cytosolic marker and DNA topoisomerase II as a nuclear marker. Left panel: the bands obtained with blot were quantified by densitometric analysis with ImageJ software. The intensity of phosphorylated RelA/p65 (S536) bands was normalized with the intensity of the controls (α-tubulin and DNA topoisomerase II). The values found for the untreated control B-cells were set as 1 and the fold change in the presence of Tat was calculated. The experiment was carried out on blood from two different healthy donors. (C) NF-κB activation is regulated by OS and Tat transcriptional activity. The Y axis shows the proportion of B-cells treated with 250 ng/ml Tat exhibiting the nuclear staining for the phosphorylated RelA/p65 compared to the untreated control, for which the obtained basal value was set as 1. A minimum of 100 cells was analyzed per condition. The experiment was carried out on blood from two different healthy donors. (D) NF-κB inhibition decreases ROS production in Tat-treated B-cells. B-cells were treated with 250 ng/ml Tat alone or with 1 mM NF-κB inhibitor BAY 11–7082. ROS production was measured by DHE staining 6 h after treatment. The Y axis shows ROS production in Tat-treated B-cells compared to the negative control, for which the obtained basal value was set as 1. A minimum of 1000 cells were examined for each condition. The experiment was carried out on blood from two different healthy donors. (E) NF-κB inhibition leads to the reduction of Tat-induced DD. The Y axis shows the proportion of γH2AX-positive B-cells treated with 250 ng/ml Tat alone or together with 1 mM NF-κB inhibitor Bay 11–7082, for 6 h, compared to the untreated controls, for which the obtained basal value was set as 1. The results were calculated from a minimum of 100 cells examined per condition. The experiment was carried out on blood from two different healthy donors. All data are expressed as the mean±SEM. The statistical analyses were carried out by the one-way ANOVA test. The statistical significance was calculated vs. untreated controls; ***: p value<0.001; **: 0.001
Article Snippet: After three washes in TBS and 0.1% Tween (5 min, RT), the membrane was incubated with antibodies against human Phospho-Rel A/NF-κB p65 (R&D Systems, 1:1000), mouse antibodies against α-tubulin (Sigma, 1:1000) or human Topoisomerase II (1:10000, Merck Millipore) for 2 h at RT with gentle rocking.
Techniques: Translocation Assay, Staining, Control, Western Blot, Incubation, Marker, Software, Activation Assay, Activity Assay, Inhibition, Negative Control
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