Figure 4
Figure 4. Slp4-a and MyRIP have opposing effects on WPB exocytosis and VWF secretion. Quantification of mRNA for Slp4-a (Ai) and MyRIP (Bi) after nucleofection with either control (siCTRL) or specific KD oligos as indicated. mRNA was depleted by 65% ± 3% (Ai) or 67% ± 3% (Bi) in KD compared with control. Data were pooled from 3 independent experiments. (Aii,Bii) Immunoblots for Slp4-a or MyRIP, respectively, after nucleofection with either control (siCTRL) or specific KD oligos as indicated. Data are representative of 3 independent experiments. Position of molecular weight markers (kDa) are shown to the left and α-tubulin used as loading control. (Aiii,Biii) Immunostaining of HUVECs for Slp4-a (rabbit Ab, green) and VWF (sheep Ab, red; Aiii) or MyRIP (goat Ab; green) and VWF (rabbit Ab, red; Biii) in control or after KD as indicated. Scale bars represent 10 μm. (Aiv,Biv) Histamine-evoked VWF secretion, quantified by ELISA, from HUVECs nucleofected with control, Slp4-a (Aiv) or MyRIP (Biv) siRNA oligos as indicated. White and gray bars represent unstimulated (vehicle alone) and histamine-stimulated conditions, respectively. Data were pooled from 3 independent experiments. A small but significant decrease in basal release was seen with Slp4-a KD (siCTRL, 0.9% ± 0.08% vs siSlp4-a, 0.57% ± 0.1% of total VWF, P = .01) and a trend to an increase in basal secretion for MyRIP KD (siCTRL, 0.27% ± 0.07% vs siMyRIP, 0.43% ± 0.15% of total VWF), although the latter did not reach significance (P = .4). (Ci) HUVECs expressing EGFP-Slp4-a (top, green) or EGFP-MyRIP (bottom, green) 24 hours after nucleofection and immunolabeled for VWF (sheep Ab; red). Grayscale images are from regions indicated by white boxes. Scale bars represent 20 μm. (Cii). Top: Fura-2 fluorescence ratio showing intracellular Ca2+ rise. Bottom: Cumulative plot of WPB fusion event times, normalized by their total number, in Proregion-EGFP- (black, 46 cells, 2151 fusion events), EGFP-Slp4-a– (red, 15 cells, 849 fusion events), and EGFP-MyRIP–expressing cells (blue, 21 cells, total absence of fusion events). Inset: The initial period after stimulation on an expanded time scale. (Ciii) Mean delays (seconds), maximal rates of exocytosis (WPBs/second), and probabilities of WPB exocytosis, Pr, (percent, note broken Y scale) for Proregion-EGFP (black) or EGFP-Slp4-a–expressing cells (red). (Civ) Relationship between the average intensity of endogenous WPB-Slp4-a-IR (gray) or endogenous WPB-MyRIP-IR (black) and WPB-EGFP FI (determined for individual WPBs) in cells expressing EGFP-MyRIP or EGFP-Slp4-a, respectively. In each cell (n = 32, EGFP-MyRIP; n = 30, EGFP-Slp4-a), at least 15 WPBs were quantified to determine the intensity of WPB-IR and EGFP fluorescence. WPB-IRs were scaled to the mean intensity of IR for each antigen in nonexpressing cells (n = 40) within the same experiment.

Slp4-a and MyRIP have opposing effects on WPB exocytosis and VWF secretion. Quantification of mRNA for Slp4-a (Ai) and MyRIP (Bi) after nucleofection with either control (siCTRL) or specific KD oligos as indicated. mRNA was depleted by 65% ± 3% (Ai) or 67% ± 3% (Bi) in KD compared with control. Data were pooled from 3 independent experiments. (Aii,Bii) Immunoblots for Slp4-a or MyRIP, respectively, after nucleofection with either control (siCTRL) or specific KD oligos as indicated. Data are representative of 3 independent experiments. Position of molecular weight markers (kDa) are shown to the left and α-tubulin used as loading control. (Aiii,Biii) Immunostaining of HUVECs for Slp4-a (rabbit Ab, green) and VWF (sheep Ab, red; Aiii) or MyRIP (goat Ab; green) and VWF (rabbit Ab, red; Biii) in control or after KD as indicated. Scale bars represent 10 μm. (Aiv,Biv) Histamine-evoked VWF secretion, quantified by ELISA, from HUVECs nucleofected with control, Slp4-a (Aiv) or MyRIP (Biv) siRNA oligos as indicated. White and gray bars represent unstimulated (vehicle alone) and histamine-stimulated conditions, respectively. Data were pooled from 3 independent experiments. A small but significant decrease in basal release was seen with Slp4-a KD (siCTRL, 0.9% ± 0.08% vs siSlp4-a, 0.57% ± 0.1% of total VWF, P = .01) and a trend to an increase in basal secretion for MyRIP KD (siCTRL, 0.27% ± 0.07% vs siMyRIP, 0.43% ± 0.15% of total VWF), although the latter did not reach significance (P = .4). (Ci) HUVECs expressing EGFP-Slp4-a (top, green) or EGFP-MyRIP (bottom, green) 24 hours after nucleofection and immunolabeled for VWF (sheep Ab; red). Grayscale images are from regions indicated by white boxes. Scale bars represent 20 μm. (Cii). Top: Fura-2 fluorescence ratio showing intracellular Ca2+ rise. Bottom: Cumulative plot of WPB fusion event times, normalized by their total number, in Proregion-EGFP- (black, 46 cells, 2151 fusion events), EGFP-Slp4-a– (red, 15 cells, 849 fusion events), and EGFP-MyRIP–expressing cells (blue, 21 cells, total absence of fusion events). Inset: The initial period after stimulation on an expanded time scale. (Ciii) Mean delays (seconds), maximal rates of exocytosis (WPBs/second), and probabilities of WPB exocytosis, Pr, (percent, note broken Y scale) for Proregion-EGFP (black) or EGFP-Slp4-a–expressing cells (red). (Civ) Relationship between the average intensity of endogenous WPB-Slp4-a-IR (gray) or endogenous WPB-MyRIP-IR (black) and WPB-EGFP FI (determined for individual WPBs) in cells expressing EGFP-MyRIP or EGFP-Slp4-a, respectively. In each cell (n = 32, EGFP-MyRIP; n = 30, EGFP-Slp4-a), at least 15 WPBs were quantified to determine the intensity of WPB-IR and EGFP fluorescence. WPB-IRs were scaled to the mean intensity of IR for each antigen in nonexpressing cells (n = 40) within the same experiment.

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