Fig. 7

Cdc42 Activates Fmnl3 to Regulate CVP Formation

(A) 293T cells transfected with the plasmid encoding either Myr-GFP or C-terminally GFP-tagged Fmnl3 N-ter mutant (Fmnl3 N-ter-GFP) together with the plasmid encoding FLAG-tagged constitutive active or dominant-negative mutants of Rho GTPases, as indicated at the top, were subjected to coimmunoprecipitation analysis as in Figure 6B. IP, immunoprecipitate; WB, western blot; TCL, total cell lysate.

(B) HUVECs transfected with the plasmid encoding mCherry (1 µg) or that encoding C-terminally FLAG-tagged Fmnl3 (Fmnl3-FLAG, 1 µg) together with empty vector (0.1 µg) or the plasmid expressing RhoA Q63L (0.1 µg), Rac1 G12V (0.1 µg), or Cdc42 Q61L (0.1 µg) were immunostained with anti-FLAG antibody (FLAG) and stained with phalloidin labeled with Alexa Fluor 488 (F-actin). Note that coexpression of Fmnl3-FLAG and Cdc42 Q61L induced the formation of thin actin fibers throughout the cells. Arrows indicate mCherry signal- or FLAG signal-positive cells.

(C) Total cellular F-actin levels, as observed in (B) were quantified as in Figure 6D. Data are expressed as percentages relative to that observed in the cells transfected with empty vector and mCherry-expressing plasmid, and shown as mean ± SEM (n = 16).

(D) Projection view of confocal z stack images of the caudal regions of 33 hpf Tg(fli1:GFP) embryos injected with control MO or fmnl3 MO together with the vehicle or MO-resistant mRNA encoding WT Fmnl3 or Fmnl3 I111D are shown as in Figure 1G.

(E) The CVP width, as observed in (D), was quantified as in Figure 1H (n ≥ 4). Error bars indicate means ± SEM.

Scale bars, 30 µm in (B) and 100 µm in (D). p < 0.05; p < 0.01; n.s., no significance. See also Figure S7.