Fig 1: Role of TLR10 in neutrophil chemotaxis. a Frames captured from live single-cell imaging of TLR10 (labeled in green) staining in human neutrophils. Neutrophils were activated with concentration gradient of LPS (1 μg/mL) attained by pipette tip diffusion and imaged for 100 min (magnification, 630; scale bar, 6 μm). b HL-60 cell line was transfected with 80 nM of TLR10 siRNA (sc-40272; Santa Cruz Biotechnology) using liposome-mediated transfection (Santa Cruz Biotechnology, 10410 Dallas, TX, USA) after obtaining 80% confluence in culture. HL-60 differentiation into neutrophils was achieved by the incubation with 1.3% DMSO for 5 days. Isolated mRNA used for quantitative real time PCR and calculated the fold change using ΔΔCt method. c Immunoblots lysates of HL60-differentiated neutrophils (2 × 106). Cells were treated with 20 nM, 40 nM, and 80 nM of TLR10 siRNA and total protein isolate was hybridized against anti-TLR10 antibody. Molecular weight is depicted on the left side of the blots. d Chemotaxis experiment was performed using Boyden chamber with control, TLR10 knockdown, LPS treated (60 min; 1 μg/mL), and TLR10 knockdown + LPS treated (60 min; 1 μg/mL). Migrated cells from at least 5 different fields were counted and tallied. One representative of two in the above experiments was shown.
Fig 2: TLR10 colocalized with TLR4 on LPS challenge. a Isolated human neutrophils (1 × 106) adhered on FBS-coated coverslips were challenged with LPS (1 μg/mL) for 60 min, 90 min, and 120 min and examined by confocal microscopy for the colocalization of TLR10 (in green) and TLR4 (in red). Merged channel indicates the overlapping signals from TLR10 and TLR4 along with nuclear stain DAPI (magnification, 630; scale bar, 5 μm). b Quantification of colocalization in terms of Pearson's coefficient analyzed by Imaris 7.4 (Bitplane Inc.) using ImarisColoc module (*p < 0.05, compared with the control, and **p < 0.05, compared with LPS 60 min, and all data shown in terms of mean ± SEM). One representative of three in the above different experiments is shown.
Fig 3: Lipid raft-mediated endocytosis of TLR10. a, b Human neutrophils (1 × 106) adhered to FBS-coated coverslips were activated by LPS (1 μg/mL). Colocalization of TLR10 (red) and early endosomal antigen, EEA1 (green), are shown in merge panel. Treatment time points were 60 min, 90 min, and 120 min (magnification, 630; scale bar, 6 μm). Lower panel shows the graphical representation of quantification of colocalization in terms of Pearson's coefficient (*p < 0.05), analyzed by Imaris 7.4 (Bitplane Inc., Concord, MA, USA) using ImarisColoc module. c, d Colocalization of TLR10 (green) and flotillin-1, lipid raft marker (red) in LPS (1 μg/mL)-treated human neutrophils (1 × 106) adhered to FBS-coated coverslips. Intact plasma membrane in control cells and membrane rearrangement during 60–120 min was observed (magnification, 630; scale bar, 6 μm). Lower panel shows the graphical representation of quantification of degree of colocalization in terms of Pearson's coefficient (*p < 0.05), analyzed by Imaris 7.4 (Bitplane Inc., USA) using ImarisColoc module. Results in (a–d) show representative data of three independent experiments.
Fig 4: TLR4 neutralization, ROS depletion, and NF-κB inhibition reduced TLR10 expression. a Isolated human neutrophils (1 × 106) treated for TLR4 neutralization and activated using bacterial LPS (1 μg/mL). TLR10 (in red) was imaged using confocal microscopy (magnification, 630; scale bar, 5 μm). b Quantification of fluorescence in terms of corrected total-cell fluorescence analyzed by Image J v1.47 (nih.gov, Bethesda, MD, USA) using grey scale intensity analysis. (*p < 0.05, compared with the control and all data shown in terms of mean ± SEM). c Human neutrophils (1 × 106) pretreated with FCCP [34], 5 μg/mL, for 1 h at 37°C to deplete ROS production. FCCP pretreated cells were challenged with LPS (1 μg/mL) for 60 min and imaged for TLR10 (in red) using confocal microscopy (magnification, 630; scale bar, 5 μm). d Isolated human neutrophils (1 × 106) treated for NF-κB inhibitor and treated using bacterial LPS. TLR10 (in green) was imaged using confocal microscopy and merged image in the third column indicates the expression and cytoplasmic localization of TLR10 in control, LPS 60-min treated as well as NF-κB inhibitor pretreated cells challenged with bacterial LPS for 60 min (magnification, 630; scale bar, 4 μm).
Fig 5: TLR10 knockdown decreased formation of pseudopodia. Control (a) and TLR10-silenced (b) HL-60-derived neutrophils (1 × 106) were challenged with 1-μM fMLP for 1 min. F-actin (165 nM) stained in green and nuclei (blue) stained with DAPI. Arrows (red) indicate pseudopodia formation (scale bar, 12 μm). c, d Control and TLR10 knockdown HL-60-derived neutrophils (1 × 106) were challenged with 1-μM fMLP for 1 min. TLR10 stained in green and nuclei in blue stained with DAPI (scale bar, 10 μm). e Difference between the cells with pseudopodia in control and TLR10 knockdown groups. HL-60-derived neutrophils (1 × 106) were challenged with 1-μM fMLP for 1 min, imaged under confocal microscope and counted 8 different fields (double-blinded counting).
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