Figure S3.

Quantification of ESC or pESC interaction with Wnt3a or control beads, and representative images of cytochalasin D or colcemid treatment. (A) Schematic depicting the three types of beads used in the experiments. (B) Representative annotated frames of a time-lapse live imaging of an ESC contacting and recruiting a Wnt3a bead though a cytoneme. Annotations refer to measurements in C, D and F, as follows: distance between the cell and the bead at the time of cytoneme-mediated contact (“Distance at contact”; X_c); time at initial cytoneme-mediated contact (“Time at contact”; t_c); distance between cell and bead 30 min after contact (“distance at contact + 30 minutes”; X_c+30; found empirically to be enough to capture the behavior of the cells after initial bead contact); time at which the bead is recruited by the cell (“Time of reaction”; t_r; only quantified for reactive interactions). Scale bar, 20 µm. (C) Quantification of the differential between distance at contact (X_c) and distance at contact + 30 min (X_c+30) expressed in micrometers, for all conditions. n ≥ 40 cells pooled from ≥3 independent experiments. (D) Quantification of the time of ESC bead initial contact with a cytoneme (t_c, in minutes). n ≥ 40 cells pooled from ≥3 independent experiments. (E) Quantification of the time of bead retention after reaction (in minutes) for all conditions. Bead retention time is only calculated for cells that react (according to Fig. 3 C). Data for ESC or pESC with Wnt3a beads are reused from Fig. 3 E. n ≥ 9 cells pooled from three experiments. F. Quantification of the reaction time, calculated as the difference between time at contact (t_c) and time at reaction (t_r) in minutes, for all conditions. Reaction time is only calculated for cells that react (according to Fig. 3 C). Data for ESC or pESC with Wnt3a beads are reused from Fig. 3 E. n ≥ 9 cells pooled from three experiments. For C–F, bars indicate mean, and error bars are SEM. Symbols indicate statistical significance calculated by one-way ANOVA with Tukey’s multiple comparison tests: ns, nonsignificant, P > 0.05; #, P = 0.051; *, P < 0.05; **, P < 0.01; ***, P < 0.001. (G) Representative images of ESCs (blue, left) or pESCs (orange, right) treated with DMSO, 0.25 µg/ml cytochalasin D (CytoD), H₂O, or 20 µg/ml colcemid (top to bottom) for 4 h, and stained with antibodies against α-tubulin (magenta) or phalloidin (F-actin, green) and DAPI (yellow). BF is brightfield. Scale bars, 20 µm.