Figure 2
From: PKM2 regulates endothelial cell junction dynamics and angiogenesis via ATP production
PKM2 silencing results in reduced vascular growth and filopodia number in the postnatal mouse retina. (A) Western blot of PKM2 and PKM1 expression in protein extracts from mouse retinas obtained 72 hours after intravitreal siRNA-injection. GAPDH is included as a loading control; n = 3 mice per condition. (B) Immunofluorescence of isolectin B4 (red, vessels), PKM2 (green), and nuclei (blue, Hoechst) in whole-mount P6 mouse retinas 72 hours after intravitreal siRNA-injection. Scale bar, 10 µm. (C) Immunofluorescence of isolectin B4 (green, vessels) in whole-mount P6 mouse retinas 72 hours after intravitreal siRNA-injection. Scale bar, 50 µm. Disconnected Erg/IB4-positive structures correspond to rests of hyaline membrane fragments. (D) Radial vascular growth in mouse retinas (P6) 72 hours after intravitreal siRNA-injection, means ± SEM, n = 8 mice per condition, ***p < 0.001 by Mann-Whitney test. (E) Vascular density in mouse retinas (P6) 72 hours after intravitreal siRNA-injection, means ± SEM, n = 4 mice per condition, ns non-significant by unpaired Student t-test. (F) Immunofluorescence ERG (white, endothelial cell nuclei) in whole-mount P6 mouse retinas 72 hours after intravitreal siRNA-injection. Scale bar, 50 µm. (G) ERG positive cells per vessel area in P6 mouse retinas 72 hours after intravitreal siRNA-injection, means ± SEM, n = 4 mice per condition, ns non-significant by unpaired Student t-test. (H) Immunofluorescence of isolectin B4 (blue, vessels), Ki67 (green, proliferation), and ERG (red, endothelial cell nuclei) in whole-mount P6 mouse retinas 72 hours after intravitreal siRNA-injection. Scale bar, 10 µm. (I) Percentage of Ki67-positive cells per total ERG-positive cells in P6 mouse retinas 72 hours after intravitreal siRNA-injection; means ± SEM, n = 4 mice per condition, ns non-significant by unpaired Student t-test. (J) Immunofluorescence of isolectin B4 (white, vessels) in whole-mount P6 mouse retinas 72 hours after intravitreal siRNA-injection. Each yellow asterisk marks one filopodia. Scale bar, 10 µm. (K) Number of filopodia per 100 μm of vascular front in P6 mouse retinas 72 hours after intravitreal siRNA-injection; means ± SEM, n = 4 mice per condition, *p < 0.05 by unpaired Student t-test. MW, molecular weight.
