A Novel Insight into Dehydroleucodine Mediated Attenuation of Pseudomonas aeruginosa Virulence Mechanism

Figure 6

Effect of DhL on P. aeruginosa induced apoptosis. J774-Eclone cells were plated in monolayer (5 × 10⁵ cells per well); once adhered they were infected with PAO1 bacteria previously grown in LB-DhL (MIC₅₀, 0.12 mg/mL) or only LB, at a multiplicity of infection of 20, and incubated for 2 hr at 37°C in a 5% CO₂ incubator. In a parallel experiment an equal number of J774-Eclone cells was subjected to 1 μM Staurosporine for positive control. Cells were then washed with PBS, stained with Hoechst dye, and subjected to fluorescence microscopy (Section 2). Five fields were randomly sampled from each experimental population, and all of the cells stained with Hoechst dye in each field were counted up to 500 in total. The total number of apoptotic cells with condensed or fragmented nuclei was determined in the five sampled regions and was expressed as follows: percentage of apoptosis per sample: number of apoptotic cells/total number of cells × 100. Each column is the mean of three individual experiments with two replicates per treatment. Significant (asterisk indicates ) decrease in apoptotic cells was noticed when infected with PAO1 grown in presence of DhL. Data represent the mean of three independent experiments ± SEM.