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. 2022 Oct 5;13:1003871. doi: 10.3389/fimmu.2022.1003871

Figure 2.

Figure 2

P-NP Mediated Modulation of Neutrophil Function In Vitro. (A) Representative quantification of uncoated or LQI-coated NP binding to neutrophils (blue) or other WBC (red). (B) FACS plots showing uncoated (middle) or LQI tetramer coated (right) Cy5-labelled PLGA NP binding to Ly6G+ neutrophils in whole blood. Left panel shows unstained control. RBC were lysed prior to FACS analysis. Cells with high side scatter (SSC) represent neutrophils. (C) PMA (50 nM) induced ROS production in control neutrophils (Cont.), neutrophils treated with empty (Empty NP) or DPI-containing P-NP (DPI NP) or with free DPI (DPI, 10 µM). (D) FACS analysis of CD11b expression in neutrophils before (Cont.) or following PMA (100 nM) stimulation. Neutrophils were preincubated with vehicle, empty or Nexinhib-20 loaded LQI tetramer coated NP#. The experiment was done several times in different combination of experimental conditions which cannot be averaged and presented with error bars. (E) Upper panel - western blot analysis showing Smad2 phosphorylation in neutrophils stimulated with TGFβ (10 ng/µl, 30 min.) in the presence of SB431542 (100 μM or 10 μM). Control neutrophils (leftmost lane) were treated with vehicle. Lower panel - western blot analysis of Smad 2/3 serving as loading control for the samples in the upper panel. (F) Western blot analysis of Smad2 phosphorylation and Smad2/3 expression in neutrophils which were pre-incubated with 6 or 0.6 μg/μl empty (1:10) NP, 6 or 0.6 μg/μl (1:10) SB431542 NP before stimulating with TGFβ (10 ng/μl, 30 min.). Control cells were treated only with TGFβ. Lower panel - western blot analysis of Smad 2/3 serving as loading control for the samples in the upper panel. (G) Ly6G+ Neutrophils isolated from 4T1-tumor bearing mice were incubated with empty P-NP, roscovitine-loaded P-NP or free Roscovitine. Subsequently cells were analyzed for Annexin-V binding to quantify apoptotic cells. (H) Effect of empty or roscovitine-containing P-NP on neutrophil numbers 4 hrs following P-NP administration. (I) Non-neutrophil Ly6G- cells isolated from 4T1-tumor bearing mice were incubated with empty P-NP, roscovitine-loaded P-NP or free Roscovitine. Subsequently cells were analyzed for Annexin-V binding to quantify apoptotic cells. The experiments were repeated at least 3 times with similar results. Error bars represent ± SEM. * p<0.05, ** p<0.01. ns, not significant. #The experiment was done several times in different combination of experimental conditions which cannot be averaged and presented with error bars.