Journal: Nature Communications
Article Title: The EYA3 tyrosine phosphatase activity promotes pulmonary vascular remodeling in pulmonary arterial hypertension
doi: 10.1038/s41467-019-12226-1
Figure Lengend Snippet: PAH-PASMC have elevated levels of EYA3 and DNA damage. a , b Western blot analyses of primary human PASMC and PAEC from normal lungs (CTL) and from the lungs of PAH patients (PAH-PASMC: L10, L85, PAH-PAEC: L89, L105). Blots were probed with anti-EYA3 antibody and quantitated using ImageJ, relative levels of EYA3 in multiple experiments (PASMC n = 5, PAEC n = 3) are shown in the bar graphs (plotted as mean ± SD; statistical significance (normal versus PAH-PASMC) was determined using two-tailed t -tests, ** P < 0.01, * P < 0.05). EYA3 levels were consistently higher in PAH-PASMC compared with normal PASMC. In contrast, EYA3 levels in PAH-PAEC were not elevated relative to normal PAEC. c – h EYA3 protein levels are elevated in plexiform lesions of lungs from PH patients. Representative serial immunohistochemical sections stained with antibodies toward α-SMA ( c , e , g ) and EYA3 ( d , f , h ). Human lung tissue explants were obtained from the Cincinnati Children’s Hospital Medical Center Transplant Service; normal human lung tissue (transplant donor, 62-year-old female; c , d ) and two patients with PH ( e , f 14-year-old female; g , h 8-year-old female) are shown. Scale bars = 50 μm. Higher magnification of the areas enclosed by red boxes in ( d ), ( f ), ( h ) shown to the right. ( i )PAH-PASMC have higher levels of γ-H2AX than normal (CTL) subjects. Cells were stained with anti-γ−H2AX antibody (red) and DAPI (blue). j Alkaline COMET assays show increased tail moments in PAH-PASMC. k Percentage of γ-H2AX foci-positive cells is elevated in PAH-PASMC compared with normal PASMC (CTL). The number of cells containing >10 γ-H2AX foci/nucleus were counted using ImageJ. The data represent the average of independent experiments. Graph shows mean ± SD. One-way ANOVA with Dunnett’s post-test was conducted. *** P < 0.001. l Tail moments in PAH-PASMC compared with normal PASMC (CTL). Tail moments calculated using OPENCOMET on > 100 cells per sample and two independent experiments. Graph shows the median with range. One-way ANOVA with Dunnett’s post-test was conducted. *** P < 0.001. Source Data are provided as a Source Data file
Article Snippet: Control (non-disease) human PAEC (Cat. No. CC-2530) and PASMC (Cat. No. CC-2581) were obtained from Lonza (Allendale, NJ).
Techniques: Western Blot, Two Tailed Test, Immunohistochemical staining, Staining
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