Fig 1: vWF secretion and the ability to generate surface strings are also compromised in KD-ATP6V0D1 human umbilical vein endothelial cells (HUVECs). Negative control (NC) and ATP6V0D1 siRNA were transfected into two groups of HUVECs, respectively. At 72 h later, one group of NC and KD-ATP6V0D1 cells was exposed to 80 nM PMA for 30 min to stimulate Weibel–Palade body secretion, and the other groups were exposed to DMSO (0.1%) instead. (A) Western blotting analysis of the detection of ATP6V0D1 knockdown in cell lysate collection. (B, E) Western blotting analysis of the detection of vWF multimer secretion in supernatant. Multimer gels were analyzed using the NIH ImageJ software. The quantification of supernatant vWF multimers was carried out based on the normalization of the ß-actin protein level of the cells in each well. n = 5, *p < 0.05,**p < 0.01. (C, D) 1% Triton X-100 was added into the culture medium of NC and KD-ATP6V0D1 HUVECs and cultured at 37°C for 1 h. Immunofluorescence images of two groups of HUVECs labeled against vWF (green) and nucleus (DAPI, blue) were shown. Scale bar, 10 µm. (F) The length of vWF strings at each group of HUVECs was measured by the NIH ImageJ software (n = 30 per group, ***p < 0.001). Data were expressed as mean ± SEM. Three independent experiments were performed.
Fig 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.
Fig 3: von Willebrand factor (vWF) secretion and the ability to generate surface strings are compromised in KD-HPS6 human umbilical vein endothelial cells (HUVECs). The negative control (NC) and HPS6 siRNA were transfected into two groups of HUVECs, respectively. At 72 h later, one group of NC and KD-HPS6 cells was exposed to 80 nM PMA for 30 min to stimulate WPB secretion, and the other groups were exposed to 0.1% DMSO instead. (A) Western blotting analysis of the detection of HPS6 knockdown in cell lysate collection. (B, E) Western blotting analysis of vWF multimer secretion in supernatant. The multimer gels were analyzed using the NIH ImageJ software. The quantification of supernatant vWF multimers was carried out based on the normalization of the ß-actin protein level of the cells in each well. n = 5, *p < 0.05,**p < 0.01. (C, D) Moreover, 1% Triton X-100 was added into the culture medium of NC and KD-HPS6 HUVECs and cultured at 37°C for 1 h. Immunofluorescence images of two groups of HUVECs labeled against vWF (green) and nucleus (DAPI, blue) were shown. Scale bar, 10 µm. (F) The length of vWF strings at each group of HUVECs was measured by the NIH ImageJ software (n = 30 per group, ***p < 0.001). Data were expressed as mean ± SEM. Three independent experiments were performed.
Fig 4: The number and the elongated shape of newly formed Weibel–Palade bodies (WPBs) are affected in ru endothelial cells (ECs). Primary endothelial cells were isolated from 4-week-old male WT and ru mice and cultured for 10 days, then 80 nM PMA was added to each dish to stimulate WPB secretion for 30 min, and the cells were fixed at 0, 2, 4, and 8 h after washing out. (A–D) (A'–D') Immunofluorescence images of primary endothelial cells at different time points labeled against vWF (green) and nucleus (DAPI, blue). Scale bar, 20 µm. The boxed square in (A–D) and (A'–D') was magnified as (a–d) and (a'-d'), respectively. Scale bar, 5 µm. (E) Quantitative analysis of the number of WPBs in each cell of WT and ru mice at different time points (n = 20, *p < 0.05, ***p < 0.001). (F) Feret’s diameter distribution of WPBs at each time point in WT and ru mice was analyzed quantitatively (WT0h: 428 WPBs, ru 0h: 616 WPBs, WT2h: 545 WPBs, ru 2h: 612 WPBs, WT4h: 921 WPBs, ru 4h: 903 WPBs, WT8h: 1,412 WPBs, and ru 8h: 692 WPBs). All the images were analyzed by the NIH ImageJ software. Data were expressed as mean ± SEM. Two independent experiments were performed.
Fig 5: Knockdown of ATP6V0D1 in human umbilical vein endothelial cells (HUVECs) phenocopies the abnormalities of Weibel–Palade bodies (WPBs) KD-HPS6 HUVECs. (A) Immunofluorescence images of negative control (NC) and ATP6V0D1 siRNA (KD-ATP6V0D1)-mediated knockdown in HUVECs labeled against vWF (green) and nucleus (DAPI, blue). Scale bar, 20 µm. The boxed squares were magnified, respectively. Scale bar, 5 µm. (B) Western blotting analysis of the detection of ATP6V0D1 knockdown. (C) Quantitative analysis of the number of WPBs per cell of NC and KD-ATP6V0D1 HUVECs (n = 30, **p < 0.01). (D) Feret’s diameter distribution of WPBs at each group of HUVECs was analyzed quantitatively (NC: 3,608 WPBs, black; KD-ATP6V0D1: 2,852 WPBs, gray). Data were expressed as mean ± SEM. Three independent experiments were performed.
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