Skip to main content
. Author manuscript; available in PMC: 2023 Jul 14.
Published in final edited form as: Sci Signal. 2023 May 23;16(786):eabo4863. doi: 10.1126/scisignal.abo4863

Fig. 1. NRP1 regulates flow-induced gene expression in endothelial cells.

Fig. 1

(A) HUVECs exposed to laminar or oscillatory flow or cultured under static conditions for 24 hours were stained for NRP1 (red and grey), F-actin (green) and DAPI (blue). Scale bar =20 μm. White arrows show the direction of flow. (B) NRP1 integrated density was measured in optical z-stacks and normalized to DAPI. Data are presented as means ± SEM. N=3 (laminar or oscillatory) or 6 (static) biological replicates per group from 3 independent experiments. *p < 0.05, **p < 0.005, ***p < 0.001 by one-way ANOVA. (C) HUVECs transfected for 72 hours with si-control or si-NRP1 and exposed to laminar flow for 24 hours or cultured under static condition were stained for NRP1 (green) and DAPI (blue). Scale bar = 20 μm. (D) NRP1 integrated density was measured in optical z-stacks, normalized to DAPI and expressed as percentage of si-control. Data are presented as means ± SEM. N = 3 biological replicates per group. (E) Representative immunoblotting for NRP1 and GAPDH of HUVECs transfected for 72 hours with si-control or si-NRP1 and exposed to static or laminar flow for 24 hours. (F) Quantification of NRP1 signal normalized to GAPDH signal expressed as fold change of static si-control. Data are presented as means ± SEM. N = 6 biological replicates per group. **p < 0.005, ***p < 0.001 by two-way ANOVA. (G, H) Heat-map of gene expression in HUVECs transfected for 72 hours with si-control or si-NRP1 exposed to laminar or oscillatory flow showing fragments per kilobase of transcript per million mapped reads (FPKM). (I-K) Volcano plots of RNA-seq transcriptomic data displaying significantly differentially expressed genes in si-NRP1-transfected HUVECs relative to si-control-transfected HUVECs cultured under static conditions (I), exposed to laminar flow (J) or oscillatory flow (K) for 24 hours. (L) Venn diagram of RNA-seq transcriptome data displaying the number of differentially expressed genes between si-control and si-NRP1 common or specific to static, laminar or oscillatory conditions. (M-O) GO analysis of biological processes differentially modulated in HUVECs transfected with si-control or si-NRP1 for 72 hours and cultured under static conditions (M) or laminar flow (N) or oscillatory flow (O) for 24 hours. For (G) to (O), N = 3 biological replicates per group.