Inhibiting SUMOylation in Rv3416 stimulated THP-1 human macrophages increases oxidative burst and nitric oxide levels.

For Panel A, THP-1 human macrophages were incubated with 25μM 2-D08 for 1h followed by stimulation with 15μg/ml Rv3416 for 1h. Oxidative burst was monitored by flow cytometry. In Panel A(a) shaded histogram (light grey) represents unstimulated cells, dotted line represents Rv3416 stimulated cells, while the thin black line depicts unstained cells. In Panel A(b) dark shaded histogram depicts cells treated with 2-D08 only, dotted line depicts Rv3416 treated cells and the thick black line represents Rv3416 stimulated cells pretreated with 2-D08. Multiple measures ANOVA was performed with 95% confidence interval. Bar chart in Panel B represent the Mean Fluorescence Intensities (MFI) of indicated groups as a mean ± SD of three independent experiments (n = 3). For Panel C THP-1 human macrophages were incubated with 25μM 2-D08 for 1h followed by stimulation with 15μg/ml Rv3416 for 24h. Nitric oxide level was monitored by Griess reagent method using spectrophotometer. ANOVA with Bonferroni’s post hoc test was performed was performed with 95% confidence interval. For Panel B, P value between groups Control and Rv3416 is P = 0.002; between group Rv3416 and Rv3416+2-D08 is P<0.0001.

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