Journal: Blood

Article Title: Netrin-4 induces lymphangiogenesis in vivo

doi: 10.1182/blood-2009-11-252338

Figure Lengend Snippet: Netrin-4 induces HMVEC-dLy proliferation, migration, tube formation and survival. (A) Mitogenic potential of different doses of Netrin-4 on lymphatic dermal human microvascular endothelial cells (HMVEC-dLys) compared with several known lymphangiogenic growth factors (FGF-2 or bFGF, HGF, VEGF-A (VA), VEGF-C (VC), and complete (CM) or basal cell (BM) culture media. Cell number was assessed using dojindo reagent 72 hours after treatment and expressed as fold increase versus control. (B) HMVEC-dLy proliferation under a single dose of Netrin-4 (500 ng/mL), FGF-2 (40 ng/mL), VEGF-A (50 ng/mL), or VEGF-C (300 ng/mL) assessed every 24 hours for 3 days. (C) Chemotactic effects of different doses of Netrin-4 and VEGF-C on HMVEC-dLys in a Boyden chamber assay. (D) HMVEC-dLy adhesion on various matrixes: Fibronectin (FN), Netrin-4, Collagen I (Col. I), and Poly-L-Lysin (PLL) at 10 μg/mL. (E) In vitro tube formation by HMVEC-dLys under different doses of Netrin-4, FGF-2 (bF), HGF, VEGF-A (VA), VEGF-C (VC), or complete media (CM). (F) Inhibition of serum deprivation–induced HMVEC-dLys apoptosis under different doses of Netrin-4, FGF-2, VEGF-A (VA), VEGF-C (VC), and complete media (CM). *P < .05.

Article Snippet: Cell culture and in vitro assays Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum.

Techniques: Migration, Control, Boyden Chamber Assay, In Vitro, Inhibition

Journal: Blood

Article Title: Netrin-4 induces lymphangiogenesis in vivo

doi: 10.1182/blood-2009-11-252338

Figure Lengend Snippet: Netrin-4 activates intracellular signaling pathways of HMVEC-dLys. Determination of the phosphorylation of Akt (Ser 473; Serine 473, panels A and D, respectively), p42/p44 (Thr 202/Tyr 204; Threonin 202/Tyrosine 204, panels B and E, respectively), and Ribosomal protein S6 (Ser 235/236; Serine 235/236, panels C and F) by Western blotting after Netrin-4 treatment of HMVEC-dLy. Experiments were performed in triplicate.

Article Snippet: Cell culture and in vitro assays Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum.

Techniques: Protein-Protein interactions, Phospho-proteomics, Western Blot

Journal: Blood

Article Title: Netrin-4 induces lymphangiogenesis in vivo

doi: 10.1182/blood-2009-11-252338

Figure Lengend Snippet: Netrin-4 induces in vitro lymphatic permeability. (A) Induction of GTP-RhoA and Rac1 by Netrin-4, VEGF-A, and VEGF-C treatment of HMVEC-dLys. (B) Stimulation of the phosphorylation of Tyrosine 416 of Src kinase family (SFK, Tyr416) and the Tyrosin 861 but not the Tyrosine 391 of focal adhesion kinase (FAK; Tyr861 and Tyr391) by Netrin-4 (500 ng/mL) and VEGF-C (500 ng/mL). (C) Measurement of the electrical resistance of the cell monolayer over 24 hours using the ECIS system or (D) immunostained using an anti–VE-cadherin antibody to visualize cell junctions (scale bar: 50 μm) of HMVEC-dLys seeded either on Fibronectin or Fibronectin plus Netrin-4. (E) Membrane fraction proteins prepared from HMVEC-dLys seeded as previously mentioned in panels C and D analyzed for ZO-1, VE-cadherin, and beta-catenin expression (equivalent loading assessed by coomassie blue staining). (F) Control, Netrin-4, VEGF-C overexpressing MCF7 tumor sections stained for the cell junction protein ZO-1 or the lymphatic marker LYVE1 (scale bar: 20 μm). Data presented in panels A through E are from 1 experiment and representative of 2 independent experiments. Pictures were taken on an Olympus IX71 microscope, at 400× magnification using a DP30BW Olympus camera and the MicroSuite Basic Edition Olympus software.

Article Snippet: Cell culture and in vitro assays Lymphatic dermal human microvascular endothelial cells (HMVEC-dLys), dermal human microvascular endothelial cells (HMVEC-ds), and human umbilical vein endothelial cells (HUVECs) were obtained from Lonza and cultured in EBM-2 supplemented with the EGM-2MV kit according to manufacturer's instructions (Lonza) for 7 passages maximum.

Techniques: In Vitro, Permeability, Phospho-proteomics, Membrane, Expressing, Staining, Control, Marker, Microscopy, Software

Journal: International Journal of Molecular Sciences

Article Title: Design and Engineering of “Green” Nanoemulsions for Enhanced Topical Delivery of Bakuchiol Achieved in a Sustainable Manner: A Novel Eco-Friendly Approach to Bioretinol

doi: 10.3390/ijms221810091

Figure Lengend Snippet: Cytotoxicity of the nanoemulsion with bakuchiol (dark grey) and control formulation with retinol (light grey) upon normal human skin cells: HaCaT keratinocytes ( a ) and NHDF fibroblasts ( b ), incubated for 24 h and 48 h. **** p < 0.0001, *** p < 0.0001, * p < 0.05.

Article Snippet: In vitro studies were performed on a normal human dermal fibroblast (NHDF, Lonza, Warsaw, Poland) cell line and immortalized human keratinocytes (HaCaT, LGC Standards, Łomianki, Poland).

Techniques: Control, Formulation, Incubation

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: SM induces human dendritic cell maturation which is further enhanced by cancer cell necroptosis (A) Schematic representation of the in vitro co-culture assay. moDCs alone (B) or in co-culture with spheroids (tumor cells and hd-fibroblasts) (D and E) were treated with the compound conditions depicted. 72 h post-treatment, cells were harvested and analyzed via flow cytometry. (B) Relative proportion of activated vs non-activated moDC treated alone. (C) TSNE plot depicting islands of phenotypically similar cells based on CD45, CD11c, HLA-DR, CD86, CD83, and PDL1 as markers. Included are the moDCs treated in monoculture, as well as the different co-cultures. Analyzed cells separate into three subpopulations: activated moDC – blue, non-activated moDC - light gray, tumor cells - black. (D) Relative proportion of tumor and moDCs in the different co-cultures. (E) Relative proportion of activated vs non-activated moDCs in co-culture. Error bars, mean ± SD of triplicate independent wells for each condition of at least 2 independent experiments using different donors. ∗∗p value <0.01, by two-tailed t-test. TNFα: 0.1 ng/mL, SM: 1 μM, zVad: 5 μM. (F) Heatmap depicting cytokines measured in the supernatant of spheroids composed of tumor cells with or without CAFs, 72 h post-treatment. Data are averages from 3 independent wells and black squares indicate levels measured below the detection limit. SM: 0.25-1 μM; TNFα: 0.1 ng/mL, zVad: 20 μM.

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: In Vitro, Co-culture Assay, Co-Culture Assay, Flow Cytometry, Two Tailed Test

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: SM modulates fibroblasts by downregulating myofibroblast-like CAF markers and upregulating pro-inflammatory soluble mediators CAF 2D monoculture was treated with increasing concentrations of SM or TGFβ. (A) Fold change in FAP and αSMA expression, analyzed via flow cytometry, 72 h post-treatment. (B) CAF confluence monitored overtime using IncuCyte. Error bars, mean ± SD of 3 independent experiments. (C) Heatmap depicting fold change in cytokine and chemokine concentration in the supernatant of treated CAFs (normalized to control). Results from 2 independent experiments. Black squares indicate levels measured below the detection limit.

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: Expressing, Flow Cytometry, Concentration Assay, Control

Journal: iScience

Article Title: Tumor microenvironment mimicking 3D models unveil the multifaceted effects of SMAC mimetics

doi: 10.1016/j.isci.2023.106381

Figure Lengend Snippet: Key resources table

Article Snippet: The human dermal fibroblasts (hd-fibroblasts - ATCC® PCS-201-010TM) were cultured in Fibroblast Basal Medium (ATCC PCS201030) supplemented 2% heat-inactivated FBS and Fibroblast Growth Kit-Serum Low (ATCC, #PCS-201-041) without HLL Supplement and rh EGF/TGTb1.

Techniques: Recombinant, Enzyme-linked Immunosorbent Assay, Software, Simple Western, High Content Screening

Journal: Nature Communications

Article Title: PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

doi: 10.1038/s41467-024-50098-2

Figure Lengend Snippet: a Average DNAm levels at CpGs across the whole genome (left panel), at LMRs ranked by the level of their PRC2 binding in hESCs (center panel) and within high-PRC2 regions (right panel) from 8 different tumor and normal samples. Data were obtained from TCGA WGBS datasets, (see Supplementary Data ). Error bars on the right show 95% confidence intervals for average DNAm across high-PRC2 LMRs for each sample. b Average DNAm levels at CpGs across the whole genome (left panel) and at LMRs ranked by the level of their PRC2 binding in hESCs (right panel) from young vs old human oligodendrocytes. (WGBS dataset, GSE107729, n = 6). c Average DNAm levels at CpGs across the whole genome (left panel) and at LMRs ranked by the level of their PRC2 binding in hESCs (middle panel), correlation between average DNAm at high-PRC2 LMRs and the number of passages (right panel, shaded area of line plot represents 95% CI) in in vitro cultured fibroblasts (GSE79798, n = 5). High-PRC2 LMRs are highlighted with dotted red boxes.

Article Snippet: Neonatal fibroblasts were purchased from Cell Applications (#106-05n) and old fibroblasts were isolated from skin tissue collected from deceased healthy individuals.

Techniques: Binding Assay, In Vitro, Cell Culture

Journal: Nature Communications

Article Title: PRC2-AgeIndex as a universal biomarker of aging and rejuvenation

doi: 10.1038/s41467-024-50098-2

Figure Lengend Snippet: a Average DNAm levels of neonatal and old passaged fibroblasts, and CD4 T-cells, at CpGs across the whole genome (left panel) and at the LMRs ranked by the level of EZH2-binding data of the same respective tissue (middle panels). Bottom panels show heatmaps of LMR rank number, ordered by EZH2 binding in their respective tissue (top heatmap) and in hESCs (bottom heatmap). Heatmaps are colored by EZH2 binding in the same respective tissue, i.e. purple/orange represents high and low-ranked neonatal/old LMRs ordered by neonatal/old fibroblasts PRC2 binding, respectively, and red/blue represent high and low-ranked CD4 T-cells LMRs ordered by CD4 T-cells PRC2 binding, respectively. Right panel shows the correlation of mean methylation of high-PRC2 LMRs against age or passage number. b Average methylation levels at LMRs ranked by PRC2 binding in hESCs for neonatal fibroblasts (left panels) and old fibroblasts (right panels). c Average DNAm levels of LMRs calculated from neonatal and old samples merged (all passages), ranked by PRC2 binding in hESCs (left panel) and correlation between methylation and age for high-PRC2 LMRs of both neonatal and old in vitro passaged fibroblasts (right panel). d Heatmap of normalized read density of the high-PRC2 neonatal/old LMRs in neonatal and old fibroblasts (passage 2). WGBS Neonatal and old fibroblasts were generated by our lab (GSE253987, n = 3 for neonatal samples (one donor) and n = 3 for old samples (one donor)), CD4 T-cell samples are the same as used in Fig. (6 samples 18–86 years old, GSE79798 and 2 samples 0 and 103 years old, GSE31263). ChIP data for fibroblast and CD4 T-cells were generated by our lab (GSE253987, two donors pooled at passage 2 for neonatal fibroblasts, three donors pooled at passage 2 for old fibroblasts, three donors pooled for CD4 T-cells). High-PRC2 LMRs are highlighted with dotted red boxes. Line plots with >3 samples plotted have a shaded area representing 95% CI.

Article Snippet: Neonatal fibroblasts were purchased from Cell Applications (#106-05n) and old fibroblasts were isolated from skin tissue collected from deceased healthy individuals.

Techniques: Binding Assay, Methylation, In Vitro, Generated

Journal: Biofabrication

Article Title: A biopsy-sized 3D skin model with a perifollicular vascular plexus enables studying immune cell trafficking in the skin

doi: 10.1088/1758-5090/ad5d1a

Figure Lengend Snippet: Characterization of the vascular plexus in 3D-SoC. (A) The COMSOL model displays the calculated range of shear stresses in the vascular pattern. The design recapitulates the physiological range of shear stress levels found in the cutaneous capillaries, venules, and arterioles. We divide the vasculature into three shear rate zones as low-shear (vertical interconnecting channels), mid-shear (two outermost channels, top and bottom) and high-shear (two innermost, horizontal channels). (B) Imaging of the vascular network seeded with GFP-HDBECs confirms uniform coverage of the microchannel walls. Scale bar: 1 mm; (C) Immunofluorescent staining of primary HDBECs in 3D-SoC with VE-cadherin (VECAD; white). Scale bar: 5 µ m; (D) confocal imaging of the 3D-SoC seeded with HDBECs perfused with both 20 kDa and 40 kDa dextran at time zero and sixty minutes allowing for comparison of the permeability characteristics. Scale bar: 2 mm; (E) the graph shows increased leakage of dextran in the model without HDBECs (acellular control) for both molecular weights. (F) Time-lapse transport data integrated into a COMSOL model enabled the estimation of the average permeability of the vasculature. The permeability values were determined to be 0.62 µ m s −1 for 20 kDa and 0.41 µ m s −1 for 40 kDa respectively (** = p < 0.01).

Article Snippet: Human dermal blood endothelial cells (HDBECs) (PromoCell #C-12211) and GFP-tagged HDBECs (Angio-Proteomie #cAP-0005GFP-PM) were cultured up to passage 3 in Microvascular EC Growth Medium (PromoCell #C-22020).

Techniques: Shear, Imaging, Staining, Comparison, Permeability, Control

Journal: Biofabrication

Article Title: A biopsy-sized 3D skin model with a perifollicular vascular plexus enables studying immune cell trafficking in the skin

doi: 10.1088/1758-5090/ad5d1a

Figure Lengend Snippet: Incorporation and real-time monitoring of circulating T cells in 3D-SoC. (A) Schematic representation of the stages of T cell infiltration into human skin. (B) Live immunofluorescent images showing the naïve T cells labelled with CellTracker (red) on HDBECs in the first 1–2 min (left panel; the round morphology resembles the tethering/rolling stage); between 2–5 min (middle panel; the spread morphology resembles the firm adhesion stage); and between 5–15 min (right panel; the morphology and location relative to ECs resembles the diapedesis stage). The first two images show the top view, and the right-most image shows a cross-section of the 3D-SoC. Scale bars: 5 µ m; (C) High magnification image capturing a T cell (red) with its lamellipodia squeezing between two endothelial cells (green), resembling the morphology of T cells in vivo during their movement through capillary walls (namely diapedesis). Scale bar: 2 µ m; (D) characterization of Th1 cells polarized from Naïve T cells in vitro through flow cytometry showing expression of both Interferon γ and TNFα. (E) Comparison of the attachment of the T cells to the shear stress analysis for naive and Th1 cell population. (F) Total percentage of naïve T cells and Th1 cells retained after 5 and 10 mins of flow. (G) Percentage of cells retained for distinct shear zones; HS: high-shear, MS: mid-shear, LS: low-shear. (* = p < 0.05, ** = p < 0.01, *** = p < 0.005).

Article Snippet: Human dermal blood endothelial cells (HDBECs) (PromoCell #C-12211) and GFP-tagged HDBECs (Angio-Proteomie #cAP-0005GFP-PM) were cultured up to passage 3 in Microvascular EC Growth Medium (PromoCell #C-22020).

Techniques: In Vivo, In Vitro, Flow Cytometry, Expressing, Comparison, Shear