Figure 4: PFKFB3^KD increases intercellular heterogeneity.

(a) Analysis of EC adhesive strength of isWT cells in isWT:isWT sprouts (first bar) and isPFKFB3^KD-FIL, isPFKFB3^KD-FIL/COR, isPFKFB3^KD-FIL/ADH and isPFKFB3^KD-ALL cells in a 1:1 isPFKFB3^KD:isWT mosaic sprout treated with DAPT. Modifying E^FIL, E^FIL/COR, E^FIL/ADH and E^ALL increased the fraction of isPFKFB3^KD cells that were classified as strongly adhesive, less motile cells (resulting in more heterogeneity in adhesion between sprout cells). Data are mean±s.e.m.; n=10; **P<0.01 and ***P<0.001 versus isWT for total number of strongly adhesive cells; #P<0.001: frequency of strongly adhesive isPFKFB3^KD mutants versus the expected frequency (50%); Student’s t-test. (b) E^FIL, E^FIL/COR, E^FIL/ADH, and E^ALL simulations of tip cell contribution in a 1:1 isPFKFB3^KD:isWT mosaic sprout treated with DAPT. The first bar shows the in vitro experimental data (ED, n=30 spheroids from 3 donors) obtained when using PFKFB3^KD:WT sprouts. Only isPFKFB3^KD-ALL cells showed different tip cell behaviour upon DAPT treatment. n=150; *P<0.05 versus ED; Fisher’s exact test. (c) In vitro EC spheroid assay using 1:1 mosaic PFKFB3^KD/GFP:WT^RED sprouts treated with DAPT or control vehicle (DMSO), showing that DAPT treatment did not change the impaired tip cell contribution of PFKFB3^KD/GFP cells. n=70 spheroids from 3 donors; NS: not significant; Fisher’s exact test. (d-f) FRAP experiment using confluent cultures of WT and PFKFB3^KD ECs expressing a VE-cadherin-GFP fusion protein. Representative fluorescence recovery curves after photobleaching (d). Quantification of VE-cadherin mobility (e) and turnover (f) for control and PFKFB3^KD ECs (n=27 (WT) and n=28 (PFKFB3^KD) from 3 donors). Data are mean±s.e.m.; NS, not significant, *P<0.05; Student’s t-test.