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. 2022 Feb 17;9:743124. doi: 10.3389/fcell.2021.743124

FIGURE 2.

FIGURE 2

The number and the elongated shape of newly formed Weibel–Palade bodies (WPBs) are affected in KD-HPS6 human umbilical vein endothelial cells (HUVECs). (A, B) Immunofluorescence images of negative control (NC; A) and HPS6 (KD-HPS6; B) siRNA-mediated knockdown in HUVECs labeled against vWF (green) and nucleus (DAPI, blue). Moreover, 80 nM PMA was added to each dish to stimulate WPB secretion for 30 min, and the cells were fixed at 2, 4, 8, and 16 h after washing out. Scale bar, 20 μm. The boxed square in (A, B) was magnified as (a, b) respectively. Scale bar, 5 μm. (C) Western blotting analysis of the detection of HPS6 knockdown. (D) Quantitative analysis of the number of WPBs per cell of NC and KD-HS6 HUVECs (n = 30, ***p < 0.001). (E) Both NC and KD-HPS6 HUVECs were exposed to 80 nM PMA for 30 min to stimulate WPB secretion, and the cells were fixed at 2, 4, 8, and 16 h, respectively, after washing out. Immunofluorescence images showed the HUVECs at different time points labeled against vWF (green) and nucleus (DAPI, blue). Scale bar, 20 μm. The boxed square was magnified, respectively. Scale bar, 5 μm. (F) Quantitative analysis of the number of WPBs per cell of NC and KD-HPS6 HUVECs (n = 20, ***p < 0.001). (G) Feret’s diameter distribution of WPBs at each time point in NC and KD-HPS6 HUVECs was analyzed quantitatively (NC2h: 700 WPBs, KD-HPS62h: 427 WPBs, NC4h: 831 WPBs, KD-HPS64h: 410 WPBs, NC8h: 812 WPBs, KD-HPS68h: 352 WPBs, NC16h: 2,816 WPBs, and KD-HPS616h: 1,862 WPBs). Data were expressed as mean ± SEM. Two independent experiments were performed.