Journal: PLOS ONE

Article Title: Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells

doi: 10.1371/journal.pone.0285597

Figure Lengend Snippet: ( A-D ) ndLECs stimulated by overnight treatment with TNFα/IFNγ take up ( A, B ) CXCL12-AF647 or ( C, D ) the ACKR3-specific chimeric chemokine CXCL11/12-Atto565 at 37°C but not at 4°C. Uptake at 37°C is abrogated by treatment with CCX771. ( A , C ) Representative histograms and ( B, D ) quantifications of 3–5 independent experiments. One-way ANOVA. ( E, F ) Scavenging of the ACKR3-specific chimeric chemokine CXCL11/12-AF647 at 37°C by unstimulated and TNFα/IFNγ stimulated human LECs is abrogated by treatment with CCX771 ( E ) Representative histograms and ( F ) quantification of 3 independent experiments. Two-way ANOVA. ( G ) AM is not able to displace CXCL11/12-AF647 in a ligand competition assay. CXCL11/12-AF647 (50nM) scavenging assay was performed in presence of exceeding concentrations of AM. Data of three independent experiments are shown as mean ±SD. Each dot represents the value obtained in an individual experiment. Statistics: RM One-way ANOVA, Dunnett’s multiple comparison test.

Article Snippet: Single donor human neonatal dermal lymphatic microvascular endothelial cells (ndLECs) were obtained from Lonza (Visp, Switzerland, catalogue #CC-2812; HMVEC-dLyNeo) and cultured in complete EBM-2 medium consisting of EBM-2 basal Medium (CC-3156, Lonza) supplemented with EGM-2 MV Single Quots TM (CC-4147, Lonza).

Techniques: Competitive Binding Assay, Comparison

Journal: PLOS ONE

Article Title: Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells

doi: 10.1371/journal.pone.0285597

Figure Lengend Snippet: ( A ) Structure of the custom-synthetized, disulfide bridged AM-AF568 according to . The unnatural amino acid L-propargyl glycine (Pra) was inserted at position 9 of the AM sequence to allow for site-specific fluorescent labeling (see Materials and methods ). ( B ) AM-AF568 induces comparable LEC proliferation as unlabeled AM. Each data point represents the mean of one experiment with 10 replicates. Mixed-effects analysis, Šídák’s multiple comparison test. ( C, D ) Uptake of either 50nM AM-AF568 or 50nM CXCL11/12-AF568 by unstimulated or TNFα/IFN γ stimulated LECs in presence or absence of CCX771 was quantified by FACS. Effect of CCX771 on uptake of ( C ) AM-AF568 and ( D ) CXCL11/12-AF647. Pooled data from 3 experiments are shown. One-way ANOVA, followed by Tukey’s multiple comparison test. ( E, F ) ndLECs were incubated with ( E ) AM-AF568 (500nM) or ( F ) CXCL11/12-AF647 (50nM) at either 4°C or 37°C and uptake into cells was analysed by confocal microscopy after 1h. Representative images from two independent experiments. Scale bars: 15μm. ( G-I ) LECs were incubated with either 50nM, 500nM or 1μM AM-AF568 in presence or absence of CCX771, and uptake was analysed by flow cytometry. ( G ) Representative histogram showing AM-AF568 internalization in stimulated LECs incubated with 50nM and 500nM. ( H, I ) Pooled data from three independent experiments performed with ( H ) unstimulated LECs and from two independent experiments performed with ( I ) TNFα/IFN γ stimulated LECs are shown. Results are shown as mean ±SD. Two-way ANOVA, followed by Tukey’s multiple comparison test. A p-value of p≥0.05 was considered not significant (ns).

Article Snippet: Single donor human neonatal dermal lymphatic microvascular endothelial cells (ndLECs) were obtained from Lonza (Visp, Switzerland, catalogue #CC-2812; HMVEC-dLyNeo) and cultured in complete EBM-2 medium consisting of EBM-2 basal Medium (CC-3156, Lonza) supplemented with EGM-2 MV Single Quots TM (CC-4147, Lonza).

Techniques: Sequencing, Labeling, Comparison, Incubation, Confocal Microscopy, Flow Cytometry

Journal: PLOS ONE

Article Title: Reassessing the adrenomedullin scavenging function of ACKR3 in lymphatic endothelial cells

doi: 10.1371/journal.pone.0285597

Figure Lengend Snippet: ( A ) Titration of AM in a LEC proliferation assay. Data from one out of two similar titration assays, performed with ndLECs and adLECs with 10 technical replicates per condition, are shown. ( B ) Treatment with CCX771 does not affect baseline proliferation of ndLECs. ( C ) CCX771 treatment does not alter AM-induced proliferation of primary human ndLECs. Data of three experiments with 10 technical replicates are shown in (B,C) and represented as mean ±SD. RM One-way ANOVA, Šídák’s multiple comparison test. ( D ) shRNA-mediated knockdown of ACKR3 does not alter proliferation induced by either 0.1nM, 1nM and 10nM AM in adLECs. Data of one out of one or two similar experiments with 10 technical replicates per condition are shown. ( E ) Data from ( D ) are shown as percentage of proliferation induced when compared to the corresponding untreated control. ( F, G) Proliferation induced by 1nM AM was investigated in shRNA-transduced jdLECs. Data of three experiments are shown and represented as mean ±SD. Each data point represents the mean of 8 technical replicates, showing ( F ) Absolute fluorescence units as a readout of proliferation or ( G ) Percentage of proliferation induction relative to the untreated control. Statistics: Two-way ANOVA, Šídák’s multiple comparison test (D,E). RM-Two-way ANOVA, Šídák’s multiple comparison test (F,G). A p-value of p≥0.05 was considered not significant (ns).

Article Snippet: Single donor human neonatal dermal lymphatic microvascular endothelial cells (ndLECs) were obtained from Lonza (Visp, Switzerland, catalogue #CC-2812; HMVEC-dLyNeo) and cultured in complete EBM-2 medium consisting of EBM-2 basal Medium (CC-3156, Lonza) supplemented with EGM-2 MV Single Quots TM (CC-4147, Lonza).

Techniques: Titration, Proliferation Assay, Comparison, shRNA, Knockdown, Control, Fluorescence

Journal: Pediatric blood & cancer

Article Title: Signaling Pathways and Inhibitors of Cells from Patients with Kaposiform Lymphangiomatosis

doi: 10.1002/pbc.27790

Figure Lengend Snippet: Cells from KLA patients and neonatal human lymphatic endothelial cells (LEC) cultured on fibronectin-coated plates. Phase contrast pictures were taken using a 10X objective, scale bar is 200 μM. Pictures of immunostaining for D2–40 (podoplanin) immunofluorescence pictures were taken using a 40X objective, scale bar is 50 μM.

Article Snippet: Normal human neonatal dermal lymphatic endothelial cells (LEC) were obtained from Lonza (catalog number CC-2812).

Techniques: Cell Culture, Immunostaining, Immunofluorescence

Journal: Pediatric blood & cancer

Article Title: Signaling Pathways and Inhibitors of Cells from Patients with Kaposiform Lymphangiomatosis

doi: 10.1002/pbc.27790

Figure Lengend Snippet: AKT and ERK phosphorylation in cells from KLA patients and neonatal human lymphatic endothelial cells (LEC). Western blot analysis was performed on cell lysates (20μg total protein) from KLA cells and LEC. Blots were probed for phosphorylated-AKT (p-Ser473 and p-Thr308), total AKT, p-PRAS40 (downstream target of AKT), total PRAS40, p-ERK1/2, total ERK1/2 and actin.

Article Snippet: Normal human neonatal dermal lymphatic endothelial cells (LEC) were obtained from Lonza (catalog number CC-2812).

Techniques: Phospho-proteomics, Western Blot

Journal: Pediatric blood & cancer

Article Title: Signaling Pathways and Inhibitors of Cells from Patients with Kaposiform Lymphangiomatosis

doi: 10.1002/pbc.27790

Figure Lengend Snippet: Proliferation of cells from KLA patients and human lymphatic endothelial cells (LEC). Cells were cultured in 20% FBS in EGM-2MV media on fibronectin-coated 96 well plates. Cell number was assessed at 0, 24, 48 and 72 hours using SRB assay. Cell numbers are expressed as Log10 of fold change in cell number compared to time 0. Data represents the results from 3 independent experiments.

Article Snippet: Normal human neonatal dermal lymphatic endothelial cells (LEC) were obtained from Lonza (catalog number CC-2812).

Techniques: Cell Culture, Sulforhodamine B Assay

Journal: Pediatric blood & cancer

Article Title: Signaling Pathways and Inhibitors of Cells from Patients with Kaposiform Lymphangiomatosis

doi: 10.1002/pbc.27790

Figure Lengend Snippet: A. Inhibition of AKT and ERK1/2 phosphorylation in KLA cells and human lymphatic endothelial cells (LEC) treated with inhibitors. Western blot analysis was performed on cell lysates (10μg total protein) from KLA cells (1607, 1528, 2360) and LEC, after treatment (1 hour) with either DMSO vehicle (control), PI3K inhibitors LY294002 (10μM) and BYL719 (10μM), AKT inhibitor ARQ092 (5μM), rapamycin (15nM) or MAPK inhibitor U0126 (10μM). Blots were probed with antibodies to phosphorylated-AKT (pSer473), total AKT, p-ERK1/2, total ERK and actin. B. KLA cells and LEC were plated on fibronectin-coated plates in EGM-2MV media. After plating (5 hours) the media was changed to media containing either DMSO vehicle (control), PI3K inhibitors LY294002 (10μM) and BYL719 (5μM), AKT inhibitor ARQ092 (5μM), rapamycin (15nM) or MAPK inhibitor U0126 (10μM). Proliferation was measured using SRB assay 72 hours after treatment with inhibitors. Data represents the results from 3 independent experiments and show percent inhibition compared to vehicle control.

Article Snippet: Normal human neonatal dermal lymphatic endothelial cells (LEC) were obtained from Lonza (catalog number CC-2812).

Techniques: Inhibition, Phospho-proteomics, Western Blot, Control, Sulforhodamine B Assay

Journal: PLoS Pathogens

Article Title: KSHV requires vCyclin to overcome replicative senescence in primary human lymphatic endothelial cells

doi: 10.1371/journal.ppat.1008634

Figure Lengend Snippet: A) Primary neonatal dermal blood and lymphatic endothelial cells were seeded at the same density and infected with KSHV at the same MOI. 48 hours post infection, cells were harvested, stained for immunofluorescence with an anti-LANA antibody, and the percentage of LANA+ cells was counted. B) BECs (closed markers) and LECs (open markers) were infected with KSHV and split 1:2 every 2–3 days, at which time the percentage of LANA+ cells were counted. Numbers indicate biological replicates of the experiment and the connected lines indicate the average of all replicates (BEC: solid line; LEC: dotted line). C) qRT-PCR of KSHV genes at 48 hpi of BECs and LECs. D) LECs were infected with WT BAC16KSHV and passaged in the presence of increasing concentrations of Ganciclovir. The relative intensity of GFP expression was measured using a Typhoon fluorescent imager and compared to untreated control cells using ImageJ software. Each of these experiments were performed at least twice.

Article Snippet: Primary neonatal human dermal microvascular blood and lymphatic endothelial cells were obtained from Lonza, generally at passage 3 (Lonza; LEC product #CC-2812; BEC product #CC-2813) and their identity was confirmed by PCR with primers that recognize BEC and LEC specific genes ( ).

Techniques: Infection, Staining, Immunofluorescence, Quantitative RT-PCR, Expressing, Control, Software