human pulmonary arterial ecs paecs  (ATCC)

Journal: PLoS ONE

Article Title: Endothelial cells are a source of Nestin expression in Pulmonary Arterial Hypertension

doi: 10.1371/journal.pone.0213890

Figure Lengend Snippet: ( A ) Co-immunofluorescence staining of PCNA and Nestin reveals multiple Nestin + PCNA + cells (arrow) in the intima of a concentric lesion from a PAH patient. In contrast, no Nestin + PCNA + cells were detected in the intima of a pulmonary artery from a control subject. Scale bars: 50 μm (overview), 25 μm (detail images). For PAH, the image on the left shows an overview of the blood vessel, whereas the images in the middle demonstrate orthogonal views of z-stacks from the area indicate by a dotted box. The image on the right is an intensity projection of the whole z-stack. ( B ) Transient Nestin overexpression in rat lung ECs 72h after adenoviral transduction (qRT-PCR). β2-microglobulin ( B2m ) was used as housekeeping gene. n = 6 per group. ( C-D ) Increased BrdU incorporation over 4 h following Nestin overexpression in rat lung ECs (72h after adenovirus inoculation). (C) Representative histograms and (D) quantification. n = 6 per group. ( E ) Transient Nestin overexpression (qRT-PCR) in human control PAECs 72h after adenovirus transduction (qRT-PCR). TATA-binding protein ( TBP ) was used as housekeeping gene. (n = 4 per group). ( F ) qRT-PCR of MKI67 (Ki-67, a proliferation marker) and CXCL12 in hPAECs 72h after AdNES or AdDL70 treatment. n = 4 per group. All data shown as mean+SEM. * P <0.05, ** P <0.01 (Mann-Whitney). ( G ) Representative Western blots demonstrating the transgenic Nestin protein expression and elevated CXCL12 protein expression in PAECs 72h after adenoviral transfection with AdNES vs. AdDL70. β-actin was used as loading control.

Article Snippet: Control human pulmonary artery ECs (PAECs) were obtained from the Pulmonary Hypertension Breakthrough Initiative (PHBI) and cultured in endothelial growth medium 2 (EGM-2, Promocell, Heidelberg, Germany).

Techniques: Immunofluorescence, Staining, Over Expression, Transduction, Quantitative RT-PCR, BrdU Incorporation Assay, Binding Assay, Marker, MANN-WHITNEY, Western Blot, Transgenic Assay, Expressing, Transfection