human aortic endothelial cells haecs  (ATCC)

Journal: bioRxiv

Article Title: Suppression of non-canonical autophagy induces endothelial and cardiac dysfunction

doi: 10.64898/2025.12.17.695030

Figure Lengend Snippet: A . Recycled VEGFR2 stained with Alexa674 (Cyan) and nuclei stained with Hoechst (Red) in human aortic endothelial cells (HAEC) RUBCN KO and RUBCN WT exposed to static or 5 dynes of laminar flow during 24h. Scale bars, 1mm. Images are representative of three independently performed experiments. B. Volume of recycled VEGFR2 per cell with each point representing the average volume for at least 3 cells per field view from HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h. IgG controls as a control for VEGFR2 antibody. Values are expressed as mean±SEM. Significance was calculated for the Volume of recycled VEGFR2 with a 2-way ANOVA followed by tukey’s multiple comparison test (Flow exposure effect: *P<0.05 ; Genotype effect: ****P<0.0001; Flow exposure Genotype effect: *P<0.05 ). C. Volume of recycled VEGFR2 per cell with each point representing the average volume for at least 3 cells per field view from murine PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3 then exposed to static or 5 dynes of laminar flow during 24h. Values are expressed as mean±SEM. Significance was calculated for the volume of VEGFR2 recycled per cell with an unpaired Student t test . *P<0.05 D. Representative immunoblots of VEGFR2 and actin in isolated surface, whole-cell lysate (WCL) and biotin unbound fractions across murine PMVEC isolated from 2-month-old Atg16l1 wt and Atg16l1 DWD ki mice then exposed to static or 5 dynes of laminar flow during 24h. E. Immunoblots of total protein S-nitrosylation in whole cell lysate from PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3, then exposed to static or 5 dynes of laminar flow during 24h. F. Total protein S-nitrosylation densitometry measured by western blot in PMVEC isolated from 2-month-old Atg16l1 wt mice n=3 and Atg16l1 DWD ki mice n=3 then exposed to static or 5 dynes of laminar flow during 24h. Values are expressed as mean±SEM. Significance was calculated for the densitometry with a 2-way ANOVA followed by tukey’s multiple comparison test. G. Nitrites measured in cell culture medium in HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h.Values are expressed as mean±SEM. Significance was calculated for the nitrites with a 2-way ANOVA followed by tukey’s multiple comparison test (Flow exposure effect: **P<0.01 ; Genotype effect: *P<0.05 ). H. Immunoblots of total VEGFR2, Y1175-phosporylated-VEGFR2, Ser1177-phosphorylated-eNOS, total eNOS and b-actin proteins in whole cells lysates of HAEC RUBCN KO and RUBCN WT (n=3 independent replicates per group) exposed to static or 5 dynes of laminar flow during 24h.

Article Snippet: Human aortic endothelial cells (HAEC) were purchased at ATCC (Cat#PCS-100-011), plated on fibronectin coated plates (1μg/cm , Corning; Cat# 354008) and maintained in EBM-2 medium (Lonza, Cat# CC-3156) completed with Endothelial Cell growth medium-2 bullet kit (Lonza, Cat# CC-3162) in a humidified 5% CO chamber at 37°C.

Techniques: Staining, Control, Comparison, Isolation, Western Blot, Cell Culture