Fig. 4: AMPK activation controls VASP phosphorylation and localisation at cell-cell junction.

a, e Western blot analysis of phosphorylated VASP (T278), total VASP and GAPDH in DMSO- and AICAR-treated astrocytes (a) and siCTL, siPTEN#1, DMSO- and VO-OHpic-treated cells (e). b, f Ratio of pVASP/VASP in DMSO- vs AICAR-treated cells (b, +31%, N = 3, two-tailed paired t-test), in siCTL vs siPTEN#1 cells (f,+43%, N = 3, two-tailed paired t-test) and in DMSO- vs VO-OHpic-treated cells (f, N = 3, two-tailed paired t-test). c Immunofluorescence images of VASP (green), N-cadherin (magenta), F-actin (cyan) and DAPA (yellow) in DMSO- and AICAR-treated cells. White rectangles area are zoomed in the top panel. Dotted-line squares in the zoomed area are further zoomed in the bottom panels. Cell-cell junctions enriched in VASP associate with ITA anchoring (white arrowheads). Note that the absence of VASP at cell-cell junctions in AICAR-treated cells is associated with the absence of ITA. d Pearson’s coefficient of colocalized junctional N-cadherin with VASP in DMSO- and AICAR-treated siCTL cells (n = 35 cells analysed from three biologically independent experiments) and in DMSO-treated siPTEN cells (n = 30 cells analysed from three biologically independent experiments). Two-tailed unpaired t-test was used to derive p values. The drop in Pearson’s coefficient highlights the loss of VASP colocalisation with junctional N-cadherin in AICAR-treated and PTEN depleted cells. g Immunofluorescence images of VASP (green), N-cadherin (magenta) and colocalized pixels between N-cadherin and VASP (white) in siCTL and siPTEN#1 cells. White rectangles area are zoomed-in. Scale bars: 10 µm. Note the disappearance of colocalized pixels in large part of cell-cell junctions in siPTEN#1 cells (yellow arrowheads). Error bars represent SD. Full scan images of the blots and source data are provided as a Source Data file.