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. 2019 Oct 21;9:15022. doi: 10.1038/s41598-019-50866-x

Figure 3.

Figure 3

PKM2 is localized at VE-cadherin junction compartment. (A) Immunofluorescence of PKM2 (green), VE-cadherin (red) and F-actin (grey) in confluent HUVECs; single color and merged images are shown. Magnified views and de-convolved images of boxed areas below. Scale bars, 10 µm and 5 µm in the magnified views. (B) RGB (red, green, blue) profile of PKM2 (green), VE-cadherin (red) and F-actin (blue) intensity at areas of the endothelial cell junction marked by the yellow lines in A. (C) Representative merged images of Hoechst (blue) and proximity ligation assay (PLA, green) for PKM2 and VE-cadherin (left) and together with F-actin (grey; right) in HUVECs. PLA of rabbit and goat IgGs is also shown (negative). Scale bar, 10 µm. (D) Immunofluorescence of PKM2 (green), VE-cadherin (red) and CD31 (grey) in confluent VE-cadherin-expressing (VEC-pos) and VE-cadherin–deficient (VEC-null) mouse endothelial cells; single color and merged images of green and red channels are shown. Scale bar, 10 µm. (E) Western blot of VE-cadherin and PKM2 in lysates from VEC-pos and VEC-null endothelial cells. Tubulin is included as a loading control. MW, molecular weight. (F) Immunofluorescence of PKM2 (green), VE-cadherin (red) and F-actin (grey) in 3D spheroid sprouts. Scale bar, 10 µm. (G) Western blot of PKM2 in total lysate and in cytosol and membrane fractions of confluent HUVECs treated with S1P or vehicle (left) and transfected with control or PKM2. VE-cadherin and Rho-GDI are included as markers of plasma membrane and cytosol. MW, molecular weight. (H) Western blot of PKM2 in membrane subfractions obtained from confluent HUVECs by density gradient centrifugation. VE-cadherin and EEA1 are included as markers of plasma membrane and endosomes. MW, molecular weight. (I) Western blot and quantification of VE-cadherin in HUVECs transfected with control or PKM2 siRNA. Tubulin is included as a loading control. Means ± SEM, n = 3 independent experiments, ns non-significant by unpaired Student t-test. (J) Immunofluorescence of VE-cadherin (red) and PKM2 (green) in siRNA-silenced confluent HUVECs. Scale bar, 10 µm. (K and L) Mean fluorescent intensity (M.F.I.) of PKM2 (K) and VE-cadherin (L) at the junctions of siRNA-silenced confluent HUVECs; means ± SEM, n = 5 independent experiments, ****p < 0.0001 by Mann-Whitney test. (M) Percentage of discontinuous VE-cadherin-junctions; means ± SEM, n = 3 independent experiments, *p < 0.05 by unpaired Student t-test. (N) Immunofluorescence of plakoglobin (red) and nuclei (blue, Hoechst) in siRNA-silenced confluent HUVECs. Scale bar, 10 µm. (O) Change in trans-endothelial electrical resistance (TEER) across monolayers of siRNA-silenced HUVECs left untreated or treated with 1 μM S1P for 3 hours; means ± SEM, n = 3 independent experiments, ns non-significant, *p < 0.05 by one-way ANOVA with Sidak post-test. See related Figure S2.