Fig. 2

Shear-sensitive NOTCH upregulates GJA4 expression. a Exposure of HUVEC to 6 h FSS significantly upregulated GJA4 protein, in parallel with sustained increase in NICD (mean densitometry ± SEM; n = 5 (Static, 3 h FSS), n = 4 (6 h FSS); one-way ANOVA: p = 0.04 (NICD), p = 0.004 (GJA4), asterisks indicate p < 0.05 in post hoc t-test vs. Static). Uncropped blots presented in Supplementary Fig. 3. b si-GJA4 and DAPT (alone or in combination) abrogated flow-induced suppression of EdU incorporation at 12–18 h shear (mean % EdU-positive nuclei ± SEM for 8 ROI; representative of n = 3 experiments; one-way ANOVA: p < 0.0001 (12 h FSS, 18 h FSS), asterisks indicate p < 0.05 in post hoc t-test). c GJA4 levels were reduced by treatment of confluent HUVEC with 8 h or 24 h DAPT (mean relative mRNA expression ± SEM vs. DMSO; n = 3 (8 h), n = 5 (24 h); individual values plotted where n < 5; Students’ t-test: p = 0.01 (8 h), p = 0.006 (24 h)). d 24 h DAPT also reduced GJA4 protein levels in confluent HUVEC. Uncropped blots presented in Supplementary Fig. 3. e Seeding of sub-confluent HUVEC onto recombinant DLL4 increased GJA4 mRNA levels (mean relative mRNA expression ± SEM vs. PBS; n = 5 for all groups; Students’ t-test: p = 0.03 (3 h), p = 0.002 (6 h), p < 0.0001 (9 h)). f GJA4 protein and NICD were increased following seeding of sub-confluent HUVEC onto Dll4 for 8 h. Uncropped blots presented in Supplementary Fig. 3. g Significant GJA4 promoter fold enrichment was detected in samples assayed by ChIP using anti-RBP-Jκ, compared to IgG controls. This effect was abolished with DAPT treatment (mean fold enrichment ± SEM vs IgG; n = 3 for all groups; Students t-test: p = 0.007 (anti-RBP-Jκ)). h si-NOTCH1, but not si-NOTCH4, abolished DLL4-induced upregulation of GJA4 mRNA at 6 h following cell seeding (mean relative mRNA expression ± SEM vs. PBS; n = 7 (si-Ctrl, si-NOTCH1), n = 8 (si-Ctrl, si-NOTCH4); Students’ t-test: p < 0.0001 (si-Ctrl), p = 0.02 (si-NOTCH4))