Figure 2.
Cortical redistribution of NMIIA during neutrophil arrest requires the LTB4–BLT1 signaling axis. (A–C) Neutrophils purified from WT mice were stained with CellTracker Green CMFDA, treated with 40 µM Y27632 or 10 µM nBleb or vehicle (DMSO) for ∼30 min, adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by 2P-IVM. Maximum intensity projections of Z-stacks are presented (A). Magenta arrowheads highlight neutrophils (green) extravasating from the blood vessels (blue). Scale bars = 20 µm. Quantification of the percentage of neutrophils that exhibited arrest (B) or extravasation (C) during the first 15–45 min after neutrophil transfer. Data are plotted as mean ± SEM from n = 3 mice for each condition, and each dot represents the result from an individual animal. An unpaired t test with Welch’s correction was used to determine statistical significance. (D–H) Neutrophils purified from GFP-NMIIA mice were treated with either 5 µM MK886 or the vehicle (DMSO), adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by ISMic. Maximum-intensity projections of Z-stacks are presented (D). Magenta arrowheads highlight extravasating GFP-NMIIA neutrophils (green) from the blood vessels (blue). Scale bars = 20 µm. Quantification of the percentage of neutrophils that exhibited arrest (E) or extravasation (F) response. Data are plotted as mean ± SEM; n = 4 mice for vehicle and n = 3 mice for the MK886 treatment; each dot represents the result from an individual animal. Unpaired t test with Welch’s correction was used to determine statistical significance. Still images in G represent an individual optical slice from a Z-stack acquired in time-lapse mode (see Video 5). GFP-NMIIA is shown in white, and blood vessels are shown in blue. Green arrowhead points to the protrusive front of the extravasating neutrophil. Orange arrowheads indicate a region of cortical NMIIA enrichment. Time is represented in min:s. Scale bars = 5 µm. The cortical redistribution of NMIIA was determined as described in the Materials and methods section and in Fig. S1 E and represented as redistribution index (H). Data points represent 70 cells from n = 4 mice for the vehicle and 51 cells from n = 3 mice for MK886 treatment, with each dot representing the value of a single cell. Unpaired t test with Welch’s correction was used to determine statistical significance. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

Cortical redistribution of NMIIA during neutrophil arrest requires the LTB4–BLT1 signaling axis. (A–C) Neutrophils purified from WT mice were stained with CellTracker Green CMFDA, treated with 40 µM Y27632 or 10 µM nBleb or vehicle (DMSO) for ∼30 min, adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by 2P-IVM. Maximum intensity projections of Z-stacks are presented (A). Magenta arrowheads highlight neutrophils (green) extravasating from the blood vessels (blue). Scale bars = 20 µm. Quantification of the percentage of neutrophils that exhibited arrest (B) or extravasation (C) during the first 15–45 min after neutrophil transfer. Data are plotted as mean ± SEM from n = 3 mice for each condition, and each dot represents the result from an individual animal. An unpaired t test with Welch’s correction was used to determine statistical significance. (D–H) Neutrophils purified from GFP-NMIIA mice were treated with either 5 µM MK886 or the vehicle (DMSO), adoptively transferred into Alox5−/− mice with inflamed footpads, and imaged by ISMic. Maximum-intensity projections of Z-stacks are presented (D). Magenta arrowheads highlight extravasating GFP-NMIIA neutrophils (green) from the blood vessels (blue). Scale bars = 20 µm. Quantification of the percentage of neutrophils that exhibited arrest (E) or extravasation (F) response. Data are plotted as mean ± SEM; n = 4 mice for vehicle and n = 3 mice for the MK886 treatment; each dot represents the result from an individual animal. Unpaired t test with Welch’s correction was used to determine statistical significance. Still images in G represent an individual optical slice from a Z-stack acquired in time-lapse mode (see Video 5). GFP-NMIIA is shown in white, and blood vessels are shown in blue. Green arrowhead points to the protrusive front of the extravasating neutrophil. Orange arrowheads indicate a region of cortical NMIIA enrichment. Time is represented in min:s. Scale bars = 5 µm. The cortical redistribution of NMIIA was determined as described in the Materials and methods section and in Fig. S1 E and represented as redistribution index (H). Data points represent 70 cells from n = 4 mice for the vehicle and 51 cells from n = 3 mice for MK886 treatment, with each dot representing the value of a single cell. Unpaired t test with Welch’s correction was used to determine statistical significance. *, P < 0.05; **, P < 0.01; ****, P < 0.0001.

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