human pulmonary microvascular ecs (hpmecs)  (Lonza)

Journal: QJM: An International Journal of Medicine

Article Title: Acute endothelial stresses identify microRNA let-7b-5p and non-coding SLC11A2 (NRAMP2/DMT1) exon as biomarkers that overlap with those detected in malignant and non-malignant diseases

doi: 10.1093/qjmed/hcae235

Figure Lengend Snippet: Pre-miRNA and mature miR responses to ferric citrate treatment of normal primary ECs. ( A ) RNA-seq data for all pre-miRNAs detected in 1 and 6 h rRNA-depleted libraries from EC treated with media or 10 μmol/l ferric citrate, presented as fold change of respective media-treated EC represented at 0 h. One hour data are from normal HDMEC compared to paired 1 h media-treated HDMEC. Six hour data are from normal HPMEC compared to paired 6 h media-treated HPMEC. By Dunn’s test post Kruskal–Wallis, there was a fall (* P < 0.05) after 1 h treatment, and no significant change after 6 h. ( B ) RNA-seq data for let-7 pre-miRNAs as in (A) (let-7a-1, let-7a-2, let-7a-3, let-7b, let-7d, let-7f-1, let-7g, let-7i), with P values (* P < 0.05) calculated by Dunn’s test post Friedman. ( C ) RNA-seq data for mRNA changes in HPMEC after 6 h 10μmol/l ferric citrate, as fold change of respective values in paired media-treated HPMEC, categorized by whether mRNAs were identified by TargetScan 5.2[28] as a let-7 target based on mature miRNAs from miRbase: 570 of 10 851 mRNAs had 1-6 (mean 1.11) let-7 family 8mer, 7mer-m8 or 7mer-1A binding sites . ( D ) Comparisons of let-7b by qRT-PCR and RNA-seq. (i) Mature miRNA let-7b-5p, assayed at stated durations of 10μmol/l ferric citrate as quantified in HPMEC by qRT-PCR using Applied Biosystems miRNA assay 002619. Ct values were converted to concentrations following spiking of cell extracts with known concentrations of cel-miR-39 as described in the . (ii) RNA-seq comparisons (as also represented in A and B). ( E ) Dose–response curves for mature let-7b-5p in four cultures of primary HDMEC or primary HUVEC, after 1 h treatment with media (‘0’), 4 or 10 μmol/l ferric citrate treatments, quantified by qRT-PCR using miRNA assay 002619 (Applied Biosystems). Threshold cycles (Ct) values were converted to concentrations based on known concentrations of spiked cel-miR-39 (described further in ), before fold-changes from the media-treated EC were calculated for graphical presentation.

Article Snippet: For treatment duration, no morphological changes were observed in primary human pulmonary microvascular ECs (HPMEC) or primary human dermal microvascular ECs (HDMEC, PromoCell GmbH, Heidelberg) cultured for 24 h with 10 μmol/l ferric citrate: 1 h and 6 h responses were evaluated.

Techniques: RNA Sequencing, Binding Assay, Quantitative RT-PCR

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Synergistic Role of Endothelial ERG and FLI1 in Mediating Pulmonary Vascular Homeostasis

doi: 10.1165/rcmb.2016-0200OC

Figure Lengend Snippet: Loss of ERG and FLI1 induces the expression of inflammatory and IFN genes in HPMECs. Human pulmonary microvascular ECs (HPMECs) were subjected to siRNA-mediated silencing of FLI1 and/or ERG for 48 hours. Scrambled siRNA (siRNA) was used as a control. Relative mRNA expression was determined by quantitative RT-PCR (n = 3 independent experiments). Data shown as mean (±SD). One-way ANOVA followed by Tukey’s multiple comparison were used for statistical analysis. *P < 0.05, **P < 0.01, ***P < 0.001. ns, not significant.

Article Snippet: Human pulmonary artery ECs (HPAECs) and human pulmonary microvascular ECs (HPMECs) were purchased from Lonza (Basel, Switzerland).

Techniques: Expressing, Control, Quantitative RT-PCR, Comparison

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Synergistic Role of Endothelial ERG and FLI1 in Mediating Pulmonary Vascular Homeostasis

doi: 10.1165/rcmb.2016-0200OC

Figure Lengend Snippet: Loss of ERG and FLI1 induces the expression of IRF proteins in HPMECs. HPMECs were subjected to siRNA-mediated silencing of FLI1 and/or ERG for 48 hours. siSCR was used as a control. Representative immunoblots and bar graphs with the densitometric analysis are shown (n = 3 independent experiments). Data shown as mean (±SD). One-way ANOVA followed by Tukey’s multiple comparison were used for statistical analysis. *P < 0.05, **P < 0.01.

Article Snippet: Human pulmonary artery ECs (HPAECs) and human pulmonary microvascular ECs (HPMECs) were purchased from Lonza (Basel, Switzerland).

Techniques: Expressing, Control, Western Blot, Comparison

Journal: American Journal of Respiratory Cell and Molecular Biology

Article Title: Synergistic Role of Endothelial ERG and FLI1 in Mediating Pulmonary Vascular Homeostasis

doi: 10.1165/rcmb.2016-0200OC

Figure Lengend Snippet: Down-regulation of ERG and FLI1 in HPMECs induces increased cell monolayer permeability. HPMECs were subjected to siRNA-mediated silencing of FLI1 and/or ERG for 48 hours. siSCR was used as a control. Fluorescence of 3 kD dextran sulfate–FITC that penetrated the endothelial layer grown on transwell inserts was measured. Vascular endothelial growth factor (VEGF; 100 ng/ml) treatment was used as a positive control. The experiment was done in triplicate. Data shown as mean (±SD). One-way ANOVA followed by Tukey’s multiple comparison were used for statistical analysis. **P < 0.01, ***P < 0.001. AU, arbitrary units; MFI, mean fluorescent intensity.

Article Snippet: Human pulmonary artery ECs (HPAECs) and human pulmonary microvascular ECs (HPMECs) were purchased from Lonza (Basel, Switzerland).

Techniques: Permeability, Control, Fluorescence, Positive Control, Comparison