Exosomes mediate LTB4 release during neutrophil chemotaxis
Fig 4
Rab27a and SMPD2 KD cells release less exosomes and show a loss in directional migration.
(A) Exosomes were purified from differentiated control (NSshRNA), Rab27a shRNA (sh1; sh3), or SMPD2 shRNA (sh2; sh4) KD cells after treatment with fMLP (2 nM, 30 min) and analyzed using a bead-based flow cytometry assay with CD63-FITC, CD81-PE, and CD11b-APC conjugated antibodies. Panels show quantitative analysis from 3 independent experiments as mean ± SD. Evaluation of exosome flow cytometry was done as described in the legend of S2A Fig. See S4B Fig for flow cytometry graphs. (B, C) Exosomes were purified from differentiated control and shRNA KD cells stimulated with 2 nM fMLP. The exosomes were lysed and their LTB4 content measured by EIA. Results from 3 independent experiments are shown as mean ± SD in pg/ml/108 cells (B) or pg/ml/μg of exosome protein (C). *** and NS indicate p < 0.0001 and p > 0.05, respectively, compared to corresponding control PLB-985 cells. (D) Differentiated control and shRNA KD cells were stimulated with subsaturating (1 nM) or saturating (1 μM) fMLP for 10 min, and the amount of LTB4 in the supernatant was assessed by EIA. Results from 3 independent experiments are shown as mean ± SD. The symbols ** and *** indicate p < 0.001 and p < 0.0001, respectively, compared to corresponding NSshRNA controls. (E) EZ-Taxiscan chemotaxis towards 1 nM of control and KD cell lines. Corresponding migration speeds and CI were calculated from 4 different experiments and represented as mean ± SD. See legend of Fig 3E for details. Also see S8 Movie. (F) Differentiated NSshRNA, Rab27a, or SMPD2 KD cells or PLB-985 cells overexpressing LTB4R1 were plated on fibronectin-coated plates for 10 min and uniformly stimulated with 1 nM fMLP. At specific time points, samples were subjected to western analyses using an antibody against pMLCII and total MLCII. Data are representative of 3 independent experiments. See S4C Fig for quantification. Raw data for panels A–D can be found in the Supporting information section S1 Data file. Uncropped blots for panel F can be found in the S1 Raw images file. CI, chemotaxis index; fMLP, N-formylMethionyl-Leucyl-Phenylalanine; KD, knockdown; LTB4, leukotriene B4; MLCII, myosin light chain II; NS, not significant; NSshRNA, nonspecific shRNA; pMLCII, phosphorylated MLCII; shRNA, small hairpin RNA.