Fig 2: Rac activation controls tubule formation with RhoG and Cdc42(a) Rac1 activation (GTP-bound Rac/total Rac) following DOCK4 depletion (si, smartpool siRNA) in HUVEC stimulated with VEGF (25 ng ml-1), bars indicate s.e.m.; n=4 independent experiments. Rac1 activation following shRNA-mediated DOCK4 depletion is shown in Supplementary Fig. 1k. (b) Images of organotypic cocultures (CD31 staining) show tubule morphology following Rho GTPase depletion (siRNA, si) in HUVEC 5 days after seeding onto CFs. Scale bar, 200 µm. Top histogram: quantification of total tubule length, number of tubules, number of branch points±s.e.m.; n=number of independent experiments (nontargeting, n=4; Rac1, n=3; RhoG, n=4; Cdc42, n=3); three cocultures quantified in each experiment. Table shows knockdown quantified by quantitative PCR. Lower histogram: branch point index. Bars represent junctions divided by tubule length±s.e.m.; n=number of independent experiments as in b. *P<0.05; **P<0.01 by two-tailed t-test compared with nontargeting control.

Fig 3: Rac GEF DOCK4 and Cdc42 GEF DOCK9 are in a complex(a) Mass spectrometry analysis following IP from 293T cells of overexpressed 3 × Flag–DOCK9 (DOCK9 interaction partners in Supplementary Table 3). (b) Upper panels show immunoblot of DOCK4 in DOCK9 or control IgG immunoprecipitated complexes (IPs) following GFP–DOCK4 and empty vector (GFP) overexpression and immunoprecipitated DOCK9 in the IPs. Lower panels show overexpressed GFP–DOCK4 and empty vector (GFP) in total lysate (TL). (c) Immunoblot of DOCK4 in DOCK9 IPs following VEGF stimulation (25 ng ml-1) and after Rac1 knockdown (Rac1 si, siRNA ol1). Fold increase of DOCK4–DOCK9 interaction compared with unstimulated control after normalization for DOCK9 in IP and DOCK4 in total lysate (TL). (d) Immunoblot of Flag-DOCK4 in DOCK9 IPs following overexpression of DOCK4 wildtype or deletion mutants in 293T shows the DOCK4-SH3 and DHR2 domains are required for interaction with DOCK9. (e) Immunoblot of DOCK9 in GST–SH3 pulldowns (PLs). Right hand panel: GST proteins visualized by Coomasie Brilliant Blue (CBB) staining. (f) Immunobots of DOCK9 (left panels) and DOCK4 (right panels) in GST–ELMO PLs following knockdown of DOCK4 and DOCK9, respectively, show DOCK4 is necessary for interaction of DOCK9 with ELMO. (g) Filopodia formation following zyxin depletion (si, siRNA smartpool) in cocultures treated with VEGF (Supplementary Fig. 1a) 5 days after seeding onto CFs visualized by CD31 staining. Arrow: tip filopodia. Scale bar, 100 µm. Histograms: quantification of total filopodia (upper panel) and lateral or tip filopodia (lower panel). Error bars s.e.m.; n=4 organotypic cocultures from two independent experiments. Knockdown was quantified by quantitative PCR. P<0.001 by two-tailed t-test compared to the indicated control.

Fig 4: A hypothetical model for the Rho-Pol II signaling shortcut in transcriptional control in cultured human cells. Once activated by extracellular signals, Rac1 and Cdc42 GTPases positively control the degradation of CTD kinases (CDK7 and CDK13) but negatively and differentially control the degradation of CTD phosphatases (FCP1 for Rac1 and RPAP2 for Cdc42) in modulation of the Pol II CTD code (Ser2P and Ser5P). Feedback regulation of DOCK4 and DOCK9, the activators of Rac1 and Cdc42, respectively, is hypothesized to be achieved by transcriptional control of these DOCK genes (indicated by dotted green arrows).

Fig 5: Rac GEF DOCK4 controls lateral remodelling and lumen formation(a) Cluster formation (arrowheads) following DOCK4 depletion (EV, empty vector; ot, on-target plus siRNA; sh, shRNA1; sp, smartpool siRNA) in HUVEC 3 days after seeding onto CFs. Scale bar, 100 µm. Scatter plot: cluster quantification, n=number of time-lapse movies (EV, n=17; non silencing, n=12; DOCK4 sh1, n=18; mock, n=6; nontargeting, n=6; DOCK4 sp, n=6; DOCK4 ot, n=6) from independent experiments (EV, n=3; DOCK4 sh1, n=3; non silencing, n=2; mock, n=2; nontargeting, n=2; DOCK4 sp, n=2; DOCK4 ot, n=2); lines represent the mean. Knockdown quantified by quantitative PCR (DOCK4 sp, ot) or immunoblot (DOCK4 sh; shown in Supplementary Fig. 1k). (b) VE-cadherin expression following DOCK4 depletion 2 days after seeding HUVEC onto CFs. (i) and (ii): magnifications of outlined boxes. Scale bar, 50 µm. (c) Branch formation (arrows) following DOCK4 depletion as in b 7 days after seeding HUVEC onto CFs. Scale bar, 100 µm. Histogram: branch point index. For each value, the number of branches divided by tubule length±s.e.m.; n=number of organotypic cocultures (EV, n=8; non silencing, n=11; DOCK4 sh1, n=8; DOCK4 sh2, n=11) from independent experiments (EV, n=3; non silencing, n=4; DOCK4 sh1, n=3; DOCK4 sh2, n=4). (d) VE-cadherin expression 7 days after seeding HUVEC with DOCK4 depletion as in b. Scale bar 25 µm. (e) Lumen formation (asterisk) following DOCK4 depletion as in c 14 days after seeding onto CFs. 3D reconstructions are from confocal Z-stacks (Supplementary Movies 1 and 2). Scale bar, 25 µm. Scatter plot: lumenized length as percentage of total length, n=number of organotypic cocultures (DOCK4 sh1, n=11; DOCK4 sh2, n=6; EV, n=15; non silencing, n=7) from independent experiments (DOCK4 sh1, n=2; DOCK4 sh2, n=2; EV, n=3; non silencing, n=2); lines represent the mean. (f) Images from Supplementary Movies 3 and 4 of tubule formation following DOCK4 depletion (shRNA1, sh; EV, empty vector) 4 days after seeding onto CFs. Arrowheads: white, protrusions persisting >5 h; red, elongating tip. Scale bar, 50 µM. Histogram: quantifications over 48 h of protrusions persisting >5 h±s.e.m.; n=number of time-lapse movies (DOCK4 sh, n=19; non silencing, n=11; EV, n=11) from independent experiments (DOCK4 sh, n=3; non silencing, n=2; EV, n=2). (g) Histogram: quantifications over 48 h of anastomoses±s.e.m.; n=number of time-lapse movies (non silencing, n=11; DOCK4 sh, n=19; EV, n=17) from independent experiments (non silencing, n=2; DOCK4 sh1, n=3; EV, n=3); NS, non significant by two-tailed t-test. Images from Supplementary Movies 5 and 6 showing anastomoses in Supplementary Fig. 1j. *P<0.05, **P<0.01, ***P<0.001; NS, non significant by two-tailed t test.