Fig 2: Paxillin activation and focal adhesion disassembly are sensitive to NRP1 disruption in ß3-integrin-heterozygous endothelial cells. (A) ECs of the indicated genotypes were seeded overnight on FN-coated glass coverslips. After 3 h of starvation, cells were treated ±VEGF for 10 min in serum-free medium, then fixed and immunostained for total paxillin (PXN; green) and neuropilin-1 (NRP1; red). Split-channel close-ups are shown to depict PXN/NRP1 colocalisation. Scale bar: 20 µm. The box and whisker plot shows Pearson's correlation coefficient of PXN/NRP1 colocalisation in each of the indicated genotypes in the indicated regions as determined using the ImageJ™ coloc2 plugin (means±interquartile ranges and extreme values; n=5 cells per genotype, over =three independent experiments). (B) Left panel: ECs of the indicated genotype were seeded overnight on FN. They were then starved for 3 h and treated ±VEGF for 10 min in serum-free medium. Cells were lysed and western blotted (WB) for levels of phosphorylated (p) and total (t) PXN. GAPDH served as a loading control. Data are representative of three independent experiments. Right panel: the graph shows densitometry of pPXN relative to tPXN, as determined by WB, over an extended VEGF time course (means±s.e.m. from =three independent experiments). (C) ECs were seeded overnight onto FN-coated glass coverslips and stained for pPXN. ECs were starved for 3 h and treated ±VEGF for 10 min in serum-free medium. Cells were fixed and immunostained for pPXN (green). Scale bar: 10 µm. (D) ECs were transfected with a PXN-GFP construct and seeded at a low density on FN-coated coverslips. 72 h later, cells were starved and then treated with VEGF in reduced-serum medium. Representative cells were then imaged live (every 2 min) on an inverted fluorescence microscope for 1 h to monitor focal adhesion (FA) remodelling. The front and back ends of individual FAs were tracked over this period to measure FA assembly and disassembly, using the ImageJ™ MTrack2 plugin. The box and whisker plot shows the rate of FA assembly or disassembly for each of the indicated genotypes (means±interquartile ranges and extreme values; n=20 FAs per genotype, from =two independent experiments). Asterisks indicate statistical significance: *P<0.05; **P<0.01; nsd, not significantly different. Unpaired two-tailed t-test.

Fig 3: Gratings enhance the establishment of FA and promote FA activation. (A,B) Representative immunofluorescence analysis of HUVECs cultured under static conditions on a flat surface and a micro-structured surface with gratings; cells were stained with (A) an antibody against paxillin (Pax, green) and (B) an antibody against phosphorylated paxillin (pPax, red). The orientation of the gratings is indicated on the panels in the lower left corner. Scale bars are 100 µm. (C) Linear density of FA analyzed in HUVECs after 3 and 17 PDs on a flat surface and a surface with gratings. The number of FAs is normalized to the cell perimeter. (D) Linear density of activated FAs. (E) FA size after 3 and 17 PDs. (F) Activated FA size after 3 and 17 PDs. Data represent the mean and standard error of the mean of 3 donors with more than 360 FAs per condition. *p < 0.05; **p = 0.01; ***p = 0.001; ns, not significant.

Fig 4: LPAR6 mediates LSD1-controlled migration. (a) Levels of PXN and PXN Y118ph. (b) Migration assay of PC-3M-luc cells treated with LPA or Ethanol (EtOH). (d) mRNA levels of LPAR6 were analysed by qRT-PCR. (e) Migration assay of PC-3M-luc cells treated with LPA or EtOH and transfected with siRNA against LPAR6 or an unrelated control siRNA. (g) Migration assay of PC-3M-luc cells treated with siRNA either against LSD1, LPAR6 or both. (h) mRNA levels of LSD1 and LPAR6 were analysed by qRT-PCR. (i) Migration assay of PC-3M-luc cells overexpressing LPAR6. (b, e, g, i) Cell indices and relative velocities are shown. (a, c, f, k) Levels of LSD1, PXN, and PXN Y118ph were analysed by western blots decorated with the indicated antibodies. ß-Actin was used as loading control. n?3. Error bars represent±s.d. or +s.d. *P?0.05, **P?0.01, ***P?0.001.

Fig 5: IR/IGF1R signaling regulates basal and glucose-stimulating insulin secretion through adhesion remodeling.A, rat primary ß-cells were cultured in 2.8 mm glucose medium for 4 h without inhibitors (CTL) or with S961 or AG1024 (some dishes were fixed corresponding to the 2.8 mm glucose condition) prior to stimulation with glucose (16.7 mm) for 20 min in the presence or absence (CTL) of S961 or AG1024. The cells were then fixed and stained for phospho-Tyr118–paxillin (pPAX in green), paxillin (PAX-red), or F-actin with Alexa 647–conjugated phalloidin (cyan). Cell area and number of big adhesions were counted for the condition of 2.8 mm glucose in the presence or absence of AG1024. (We defined a big adhesion as an adhesion with its length and width larger than 2 and 0.5 µm, respectively (n = 6). Scale bars, 10 µm. B, rat primary ß-cells were incubated in 2.8 mm glucose for 4 h alone or treated with AG1024 and then lysed or followed by stimulation in 16.7 mm glucose-containing medium for 20 min. Cell lysates were immunoprecipitated with anti-FAK antibodies. Then total cell lysates, immunoprecipitations (IP), and immunoprecipitation supernatants (S) were run on SDS-PAGE and analyzed by Western blotting. The presented blot is representative of four independent experiments. C, MIN6B1 cells were treated with AG1024 or not (CTL) for 4 h in the presence of 2.8 mm glucose. Representative Western blots stained against the respective proteins were quantified. The ratio of phospho-IR–IGF1R and total IGF1R (n = 6), phospho-AKT and total AKT (n = 6), and phospho-ERK1/2 and total ERK 1/2 (n = 7) were performed for each condition and compared against 2.8 mm glucose condition without inhibitors (fold over CTL).