Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC (A) and PASMC (B) attained confluence in low (0.2%) serum as determined by light microscopy and the absence of proliferation. Cells were then exposed to increasing concentrations of serum and cell number determined in triplicate seven days later. (n = 3 experiments; * indicates p<.05 compared with starting cell number).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Light Microscopy

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and cell cycle profile (A and C) and BrdU incorporation (B and D) determined 24 hours later. (n = 3 experiments; * indicates p<.05).

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: BrdU Incorporation Assay

Journal: PLoS ONE

Article Title: Serum Can Overcome Contact Inhibition in Confluent Human Pulmonary Artery Smooth Muscle Cells

doi: 10.1371/journal.pone.0071490

Figure Lengend Snippet: A and B) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were then exposed to 0.2% or 5% serum and 24 hours later, cell lysates were harvested for protein analysis. Representative Western blots are shown. C) Human PAEC and PASMC attained confluence in low (0.2%) serum and were maintained in low serum for seven days. Cells were infected for 2 hours at a multiple of infectivity of 200 with a replication-deficient adenovirus serotype 5 containing either a human p27 KIP1 ( Ad p27) or alkaline phosphatase ( Ad C) cDNA driven by a CMV promoter. Cells were then exposed to 5% serum and cell lysates harvested 24 hours later. A representative Western blot is shown. [pRB: hypophosphorylated retinoblastoma; ppRB: hyperphosphorylated retinoblastoma]; D) BrdU incorporation 24 hours after exposure to 5% serum in p27 KIP1 -infected human PAEC and PASMC.

Article Snippet: Human Pulmonary Artery Endothelial cells (PAEC) and Human Pulmonary Artery Smooth Muscle cells (PASMC) were purchased from Cambrex (Walkersville, MD) and used at passage 4 to 7.

Techniques: Western Blot, Infection, BrdU Incorporation Assay

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Upregulation of TMEM16A in human PAECs. ( a ) Immunofluorescence staining of TMEM16A in PAECs infected with Ctrl Ad or Ano1 Ad (BP = antibody blocking peptide; scale bar = 20 µm). ( b ) Representative whole-cell I ClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ctrl Ad . ( c ) Representative whole-cell I ClCa traces (left) and normalized current-voltage relationships (right) measured with voltage-clamp showing the effect of Bbr in donor PAECs transfected with Ano1 Ad and overexpressing TMEM16A. ( d ) Consecutive, calculated Bbr-sensitive current comparing primary PAECs infected with Ctrl Ad or Ano1 Ad . Figures were generated with 8–13 cells from N = 2 healthy donors, data are presented as mean ± s.e.m.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Immunofluorescence, Staining, Infection, Blocking Assay, Transfection, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: TMEM16A-mediated membrane depolarization disrupts Ca 2+ dynamics of human PAECs. ( a ) Fluorometric measurements indicating shift in relative resting membrane potential (E m ) of donor PAECs infected with Ctrl Ad or Ano1 Ad using DiBAC 4 (3) dye. ( b ) Representative traces depict changes in intracellular Ca 2+ detected in PAECs transfected with Ctrl Ad or Ano1 Ad . ( c – e ) The effect of TMEM16A overexpression on cytosolic baseline Ca 2+ concentration ([Ca 2+ ] i ), store depletion and Ca 2+ influx using Fura-2 in donor PAECs infected with Ctrl Ad or Ano1 Ad (BHQ = butylhydroquinone). Figures were generated with 80-116 cells from N = 3 healthy donors. * p < 0.05, *** p < 0.001, paired ( a ) and unpaired t-tests ( c – e ).

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Infection, Transfection, Over Expression, Concentration Assay, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Enhanced TMEM16A mediates metabolic changes of PAECs. ( a – b ) Seahorse mitochondrial stress test profiles of TMEM16A-overexpressing primary PAECs showing the ratio of oxygen consumption rate (OCR) to extracellular acidification rate (ECAR) OCR/ECAR. ( c ) Proliferation of human PAECs overexpressing TMEM16A measured with thymidine incorporation ( n = 5). ( d , e ) Cas3/Cas7 apoptosis assay and cell-cycle analysis of human PAECs overexpressing TMEM16A. (STS = staurosporin). ( f ) Western blots of PAECs infected with TMEM16A-overexpressing Ano1 Ad or control Ctrl Ad with quantifications of PCNA, cleaved PARP/PARP and Cyclin D1. Figures were generated with 13 separate sets of experiments. * p < 0.05, ratio-paired ( a ) or paired t-test.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Apoptosis Assay, Cell Cycle Assay, Western Blot, Infection, Generated

Journal: Cells

Article Title: Endothelial Dysfunction Following Enhanced TMEM16A Activity in Human Pulmonary Arteries

doi: 10.3390/cells9091984

Figure Lengend Snippet: Elevated TMEM16A activity disturbs eNOS activation: ( a ) Noninduced nitric oxide levels and ACh-induced nitric oxide production of control (Ctrl Ad ) and TMEM16A-overexpressing (Ano1 Ad ) human PAECs. Figures were generated with 8 sets of experiments with quadruplicate in each group and normalized to protein content. ( b ) Western blots showing ACh-induced changes in eNOS phosphorylation of Ctrl Ad and Ano1 Ad -infected donor PAECs with quantification following the eNOS phosphorylation pattern at activatory Ser1177 and inhibitory Thr495 sites after 5, 15 and 30 min of ACh stimulation. ( c ) Quantification of basal, noninduced level of eNOS phosphorylation at Ser1177 and Thr495 as well as phosphorylation of Ser1177 15 min after ACh stimulation. Figures were generated with 6 samples. * p < 0.05, *** p < 0.001, ratio-paired t-test.

Article Snippet: Human PAECs purchased (Lonza, Basel, Switzerland) or isolated as described above, were cultured in gelatin-(0.1% gelatin solution in PBS) coated cell culture flasks in Lonza endothelial cell growth medium (EBMTM-2 supplemented with EGMTM-2 containing growth factors, cytokines and other supplements, with final 2% FCS concentration), here referred to as complete medium.

Techniques: Activity Assay, Activation Assay, Generated, Western Blot, Infection