Journal: Frontiers in Cell and Developmental Biology

Article Title: VE-PTP controls a fluid shear stress set point that governs cell morphological responses through Tie-2

doi: 10.3389/fcell.2025.1603517

Figure Lengend Snippet: VE-PTP inhibition increases shear stress induced cellular morphological responses in a Tie-2-FOXO1 dependent way. (A–C) Effect of Tie-2 siRNA on AKB-9778-mediated promotion of cell alignment and elongation in 5 dyn/cm 2 of shear stress. HUVECs transfected with control or Tie-2 siRNA were exposed to shear stress for 24 h with or without AKB-9778 in EBM-2 flow medium. The resulting cells were stained for VE-cadherin (green) and VE-PTP (red) (A) . Percentage of parallel cell alignment (B) and elongation (C) were quantified with Fiji/ImageJ. (D) siRNA-mediated Tie-2 silencing efficiency. HUVECs transfected with control or Tie-2 siRNA were treated with AKB-9778 for 4 hours. The resulting cells were lysed and immunoblotted with antibodies against Tie-2, VE-PTP and α-Tubulin. (E–G) Effect of FoxO1 siRNA on VE-PTP siRNA-mediated promotion of cell alignment and elongation in 5 dyn/cm 2 of shear stress. HUVECs transfected with control, VE-PTP or FoxO1 siRNA were exposed to shear stress for 24 h in EBM-2 flow medium. The resulting cells were stained for VE-cadherin (green), FoxO1 (red) and Hoechst (blue) (E) . Percentage of parallel cell alignment (F) and elongation (G) were quantified with Fiji/ImageJ. (H) siRNA-mediated FoxO1 and VE-PTP silencing efficiency. HUVECs were transfected with control, VE-PTP or FoxO1 siRNA. The resulting cells were lysed and immunoblotted with antibodies against FoxO1, VE-PTP and α-Tubulin. Mean ± SEM; n = 7 (A–C) or 4 (E–G) ; P values are calculated with two-way ANOVA followed by Dunnett’s multiple tests ( (C) ; vs. control siRNA + AKB-9778 and (F) ; vs. VE-PTP siRNA). Scale bars: 50 μm (A,E) .

Article Snippet: The following antibodies were used for immunofluorescence and immunoblotting: Rabbit monoclonal anti-human FoxO1 (clone C29H4 #2880, Cell Signaling, 1:1,000 for immunoblotting and 1:200 for immunofluorescence staining); rabbit monoclonal anti-human LC3B (clone D11 #3868, Cell Signaling, 1:1,000 for immunoblotting); mouse monoclonal anti-human Tie-2 (clone Ab33, #05-584, Sigma, 1 μg/mL for immunoblotting or 5 μg/mL for immunoprecipitation); mouse monoclonal anti-human α-tubulin (clone B-5-1-2 #T6074, Sigma, 0.5 μg/mL for immunoblotting); mouse monoclonal anti-phosphotyrosine (clone 4G10, #05-321, Sigma, 0.5 μg/mL for immunoblotting); mouse monoclonal anti-human VE-cadherin (clone F-8 #sc-9989, Santa Cruz Biotechnology, 1:100 for immunofluorescence staining); rabbit polyclonal anti-human VEGFR2-pY1054/59 (#44-1047G, invitrogen, 1:250 for immunofluorescence staining) rabbit polyclonal anti-human VE-PTP (VE-PTPh1-8, homemade , 10 μg/mL for immunofluorescence staining); rabbit polyclonal anti-human VE-PTP (VE-PTP-C, homemade , 1 μg/mL for immunoblotting).

Techniques: Inhibition, Shear, Transfection, Control, Staining