Figure 1

PKM2 is required for in vitro endothelial cell sprouting. (A) Western blot of PKM2 and PKM1 expression 72 hours after siRNA silencing in HUVECs and quantification versus tubulin included as a loading control; means ± SEM, n = 3, ns non-significant, **p < 0.01 by unpaired Student t-test. (B) Bright-field microscopy images of spheroids coated with HUVECs transfected with control or PKM2 siRNA and embedded in fibrin gels for 7 days. Scale bar, 10 µm. (C) Sprout length in 3D spheroids; means ± SEM, n = 103 and 38 spheroids formed by control and PKM2 siRNA-silenced cells from one representative experiment of five performed, ****p < 0.0001 by unpaired Student t-test. (D) Sprout numbers in 3D spheroids; means ± SEM, n = 27 and 14 spheroids formed by control and PKM2 siRNA-silenced cells from one experiment representative of five performed, **p < 0.01 by unpaired Student t-test. (E) Immunofluorescence of Ki67 (red, proliferation) and Hoechst (blue, nuclei) in 3D spheroid sprouts. Scale bar, 10 µm. (F) Percentage of Ki67-positive cells per sprout in 3D spheroids; means ± SEM, n = 3 independent experiments, ns non-significant by paired Student t-test. (G) Immunofluorescence of F-actin in 3D spheroid sprouts. Scale bar, 10 µm. (H) Filopodia number in 3D spheroids; means ± SEM, n = 13 and 15 filopodia in sprouts formed by control and PKM2 siRNA-silenced cells from one representative experiment of five performed, ***p < 0.0001 by Welch´s test. MW, molecular weight. See also Figure S1.