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. 2021 Oct 26;22(21):11560. doi: 10.3390/ijms222111560

Figure 2.

Figure 2

PTX-dependent inhibition of HP-NAP-induced ROS production in neutrophils and ATRA-induced differentiated HL-60 cells. (A) The inhibitory effect of PTX on HP-NAP-induced ROS production by neutrophils. Neutrophils at a density of 2 × 106 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 3 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for the indicated time. ROS produced by neutrophils was measured by H2DCF-DA-derived fluorescence assay as described in Materials and Methods. Data from neutrophils treated with HP-NAP for 1.5 h are shown as bar graph and expressed as mean ± SD of four independent experiments in triplicate. (B) The inhibitory effect of PTX on the number of ROS-producing cells in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-differentiated HL-60 cells at a density of 4 × 106 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. The number of ROS-producing cells was determined by NBT reduction assay as described in Figure 1A. Data are expressed as mean ± SD of six independent experiments. (C) The inhibitory effect of PTX on the level of ROS production in ATRA-induced differentiated HL-60 cells induced by HP-NAP. ATRA-induced differentiated HL-60 cells at a density of 4 × 106 cells/mL were pretreated with 100 ng/mL PTX or the vehicle control at 37 °C for 16 h and followed by the stimulation with 1 μM HP-NAP or D-PBS, pH 7.2, as the unstimulated control at 37 °C for 30 min. The level of ROS production was measured by monitoring the cell-derived DHE fluorescence using flow cytometry as described in Materials and Methods. The representative histogram is shown in the left panel. Data in the right panel are expressed as the fold change of the mean fluorescence intensity (MFI) by defining the MFI of DHE-derived fluorescence in the unstimulated control cells as 1 and as mean ± S.D. of three independent experiments. *: p value < 0.05, ***: p value < 0.001 as compared with unstimulated cells. #: p value < 0.05, ###: p value < 0.001 as compared with HP-NAP-stimulated cells.