Figure 4

PKM2 silencing leads to misorientation and impaired collective migration of ECs. (A) Immunofluorescence of PKM2 (green) and Ki67 (red, proliferation) in siRNA-silenced HUVECs 16 hours after scratch wounding. Scale bar, 50 µm. W, wound. (B) Western blot and quantification of PKM2 expression in silenced-HUVEC cells at the end of the scratch assay; means ± SEM, n = 3 independent experiments, *p < 0.05 by Mann-Whitney test. MW, molecular weight (C and D) Percentage of wound closure (C) and of Ki67-positive cells (D) 16 hours after scratch wounding; means ± SEM, n = 3 independent experiments, ns non-significant and **p < 0.01 by unpaired Student t-test. (E) Immunofluorescence of VE-cadherin (green), PKM2 (blue), and GM130 (red) in siRNA-silenced HUVECs 16 hours after scratch wounding. W, wound. White and red arrows indicate cell polarization toward and away from the wound, respectively; Scale bar, 10 µm. (F and G) Percentage of discontinuous junctions (F) and of endothelial cells oriented against the wound (G) in siRNA-silenced HUVECs 16 hours after scratch wounding; means ± SEM, n = 3 independent experiments, **p < 0.01 by unpaired Student t-test. (H) Single-cell track analysis of siRNA-silenced HUVECs in the scratch assay; n = 3 independent experiments. Black and red tracks indicate cells migrating toward and against the wound. (I and J) Velocity, linear (Euclidean) distance and directionality of single cells moving against (I) and toward the wound (J); means ± SEM, n = 50–60 cells from 3 independent experiments, ns non-significant and *p < 0.05 by Mann-Whitney test. (K) Representative single-cell tracks of three adjacent siRNA-silenced ECs migrating during the scratch assay. See related Figure S3 and Movies S1 and S2.