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E. Karampini et al.
mEGFP in LVX-mEGFP-LIC by subcloning between the BsrGI and NotI sites. All constructs were sequence verified. Lentiviral plasmids were produced in Stbl3 bacteria.
Further details on materials and methods are provided in the Online Supplementary Data.
Results
Weibel-Palade body length is significantly reduced upon Sec22b silencing
To determine the role of longin-SNARE in WPB biogen- esis we performed a targeted shRNA screen against VAMP7, YKT6 and Sec22b in HUVEC and evaluated WPB
morphology by VWF immunostaining. As shown in Figure 1A, in shCTRL, shVAMP7 and shYKT6 transduced cells, VWF was primarily stored in typical cigar-shaped WPB. However, upon Sec22b silencing (shSec22b) (Online Supplementary Figure S1), WPB lost their characteristic elongated morphology and appeared short and “stubby”. Quantification of WPB length in control and knockdown (KD) cells showed a significant reduction in WPB length after Sec22b KD, whereas no difference was found in the absence of VAMP7 or YKT6 (Figure 1B). To further sub- stantiate the role of Sec22b in WPB formation we used CRISPR-mediated SEC22B-editing of HUVEC (Online Supplementary Figure S2A-C). Cells depleted of Sec22b were identified by Sec22b staining (Online Supplementary
AB
CDE
Figure 1. Sec22b depletion and fusogenic function affects Weibel-Palade body elongation. (A) von Willebrand factor (VWF) immunostaining in endothelial cells (EC) transduced with shCTRL, shVAMP7, shYKT6 or shSec22b (green channel: mEGFP-expressing EC). (B) Quantification of Weibel-Palade body (WPB) length in shCTRL, shVAMP7, shYKT6 and shSec22b EC (n=3, one-way analysis of variance [ANOVA] with the Dunnett multiple comparisons test, ****P<0.0001). (C) WPB length in control and CRISPR SEC22B knockout EC (n=3, one-way ANOVA with the Dunnett multiple comparisons test, ****P<0.0001). (D) VWF immunostaining in mEGFP and mEGFP-Sec22b-DSNARE expressing EC (both in green). (E) WPB length in mEGFP and mEGFP-Sec22b-DSNARE expressing EC (n=3, t-test with Welch correction, ****P<0.0001).
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haematologica | 2021; 106(4)