Figure 6

PKM2 activity is required for ATP levels at EC junctions. (A) Extracellular acidification ratio (ECAR) in HUVECs transfected with control or PKM2 siRNA; means ± SEM, n = 3 independent experiments.**p < 0.01 by Mann-Whitney t-test. (B) Total ATP per ng protein in HUVECs transfected with control or PKM2 siRNA, quantified as fold induction (F.I.) versus control; means ± SEM, n = 8 independent samples analyzed in 2 sets of experiments. *p < 0.05 by Mann-Whitney t-test. (C and D) Baseline (C) and maximum (D) oxygen consumption ratio (OCR) in HUVECs transfected with control or PKM2 siRNA; means ± SEM, n = 3 independent experiments, ns non-significant by Mann-Whitney t-test. (E) Immunofluorescence of PKM2 (green), VE-cadherin (red) and F-actin (grey) in HUVECs permeabilized or not with 0.01% saponin. Merged image of green and red channels is also shown. Scale bar, 10 µm. (F) Western blot of PKM2 in fractions extracted from HUVECs with 0.01% saponin (cytosol enriched) or 0.01% + 0.1% saponin (cytoskeleton (CSK)-junction enriched). VE-cadherin and β-actin are included as markers of cell junctions and cytoskeleton. MW, molecular weight. (G and H) Pyruvate kinase activity (G) and ATP amount (H) normalized to protein content in fractions extracted as in F from HUVECs transfected with control or PKM2 siRNA; means ± SEM, n = 8 independent samples analyzed in 2 sets of experiments, ns non-significant, *p < 0.05 and **p < 0.001 by unpaired Student t-test. (I) Images of the GFP, OFP and OFP/GFP channels from live microscopy of GO-ATeam1-transduced HUVECs silenced with control or PKM2 siRNA. The yellow and white lines mark in the GFP channel the cytosol and EC junction areas for OFP/GFP quantification. Scale bar, 10 µm. (J) OFP/GFP intensity ratio at cytosol, EC junctions and EC junction/cytosol of GO-ATeam1-transduced HUVECs transfected with control or PKM2 siRNA as fold induction respect control siRNA cells in each experiment, means ± SEM, n = 100–109 cells from 5 independent experiments analyzed per condition, ****p < 0.0001 by unpaired Student t-test. See related Figures S4, S5 and S6.