Figure 1.
PIEZO1 mediates leukocyte transendothelial migration in vitro. (A) HUVECs pretreated with 10 ng/mL TNFα were transfected with 360 siRNAs pools against RNAs encoding transmembrane proteins expressed in endothelial cells and were then exposed to THP-1 monocytic cells for 3 hours. Shown is the ratio of THP-1 cells that transmigrated the HUVEC monolayer transfected with a particular siRNA pool and with control siRNA. The plot shows the ranked average ratios of 3 independent experiments. (B) HUVECs were transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1), and rolling, adhesion, and transmigration of human PMNs applied together with flow (1.2 dynes/cm2) were analyzed (n = 8 independent experiments per group). Cells treated with control siRNA were set as 100%. (C-I) The indicated endothelial cells were transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) or were left untransfected (E). (C) Rolling, adhesion, and transmigration of mouse PMNs (n = 8 per group) applied together with flow (1.2 dynes/cm2) to a bEnd.3 cell monolayer. Cells treated with control siRNA were set as 100%. (D,F,G) Transmigration of human peripheral blood mononuclear cells (D) (n = 4 independent experiments per group), human PMNs (F) (n = 6 independent experiments per group), or mouse PMNs (G) (n = 6 independent experiments per group) across HUVECs (D,F) or bEnd.3 cells (G) pretreated without or with 1 μM Yoda1 for 15 minutes. (E) MLECs were isolated from EC-Piezo1-KO and control mice, and transmigration of mouse PMNs was determined after pretreament without or with 1 μM Yoda1 for 15 minutes (n = 5 independent experiments). (H) HUVEC barrier integrity was assessed using an electric cell-substrate impedance sensing (ECIS) system in the absence or presence of 1 μM Yoda1 (n = 8 independent experiments per group). (I) Paracellular permeability of the endothelial monolayer cultured in transwell plates was determined using 40 kDa FITC-dextran (n = 5 independent experiments per group; a.u., arbitrary units). Shown are mean values ± SEM; *P ≤ .05; **P ≤ .01; ***P ≤ .001 (unpaired t test [B-H], 2-way ANOVA [I]).

PIEZO1 mediates leukocyte transendothelial migration in vitro. (A) HUVECs pretreated with 10 ng/mL TNFα were transfected with 360 siRNAs pools against RNAs encoding transmembrane proteins expressed in endothelial cells and were then exposed to THP-1 monocytic cells for 3 hours. Shown is the ratio of THP-1 cells that transmigrated the HUVEC monolayer transfected with a particular siRNA pool and with control siRNA. The plot shows the ranked average ratios of 3 independent experiments. (B) HUVECs were transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1), and rolling, adhesion, and transmigration of human PMNs applied together with flow (1.2 dynes/cm2) were analyzed (n = 8 independent experiments per group). Cells treated with control siRNA were set as 100%. (C-I) The indicated endothelial cells were transfected with control (siCtrl) or PIEZO1-specific siRNA (siPIEZO1) or were left untransfected (E). (C) Rolling, adhesion, and transmigration of mouse PMNs (n = 8 per group) applied together with flow (1.2 dynes/cm2) to a bEnd.3 cell monolayer. Cells treated with control siRNA were set as 100%. (D,F,G) Transmigration of human peripheral blood mononuclear cells (D) (n = 4 independent experiments per group), human PMNs (F) (n = 6 independent experiments per group), or mouse PMNs (G) (n = 6 independent experiments per group) across HUVECs (D,F) or bEnd.3 cells (G) pretreated without or with 1 μM Yoda1 for 15 minutes. (E) MLECs were isolated from EC-Piezo1-KO and control mice, and transmigration of mouse PMNs was determined after pretreament without or with 1 μM Yoda1 for 15 minutes (n = 5 independent experiments). (H) HUVEC barrier integrity was assessed using an electric cell-substrate impedance sensing (ECIS) system in the absence or presence of 1 μM Yoda1 (n = 8 independent experiments per group). (I) Paracellular permeability of the endothelial monolayer cultured in transwell plates was determined using 40 kDa FITC-dextran (n = 5 independent experiments per group; a.u., arbitrary units). Shown are mean values ± SEM; *P ≤ .05; **P ≤ .01; ***P ≤ .001 (unpaired t test [B-H], 2-way ANOVA [I]).

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