Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) A schematic of an organotypic 3D lymphatic vessel model (LV-on-chip). Prox-1 (green) and CD31 (red) expression confirms lymphatic endothelial identity and cell morphology in the channel. (B) Morphologic changes in human dermal microvascular blood endothelial cells (BECs) with lymphatic endothelial cells (LECs) after one day of cell seeding. BECs become more contractile than LECs, forming a smaller vessel diameter compared to LECs. (C) BVs and LVs observed in mouse ear tissues. mLYVE-1, anti-mouse LYVE-1 antibody; mCD31, anti-mouse CD31 antibody. (D) Phalloidin (red) and anti-VE-cad (VE-cadherin) antibody (green) staining to visualize F-actin and adherens junctions. (E) Lymphatic and blood vessel barrier function. 70 kDa dextran was introduced into the vessel lumens and dextran diffusion was observed in real time under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (F) Quantification of the permeability of BEC-generated engineered BVs and LEC-generated LVs. ** p = 0.0016, two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Expressing, Staining, Diffusion-based Assay, Microscopy, Permeability, Generated, Two Tailed Test

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Lymphatic endothelial cells (LECs) in different ECM hydrogels (2D): 2.5 mg/ml collagen 1, 2.5 mg/ml collagen 1 and 150 μg/ml Fibronectin, and no gel (plastic). F-actin and VE-cad were visualized to assess cytoskeletal arrangement and adherens junction formation in each condition. (B) Quantification of the relative junction area was performed, illustrating a significantly lower junction area in cells grown on the 2.5 mg/ml collagen 1 compared to the cells grown directly on plastic. ** p = 0.0017 (Collagen 1 vs. plastic); higher junction area in cells grown on the 2.5 mg/ml collagen 1 + fibronectin compared to the cells grown on collagen 1. * p = 0.0151 (Collagen 1 + fibronectin vs. Collagen 1); not-significant (ns) p = 0.5292 (Collagen 1 + fibronectin vs plastic). One-way ANOVA with Tukey’s HSD tests , n = 6 per group. Data are expressed as mean ± S.E.M. (C) Dynamics of fibronectin on LECs in collagen 1 or collagen 1 + fibronectin gel. On collagen 1 gel, LEC islands with VE-cad expression lacks fibronectin expression. On collagen 1 + fibronectin, fibronectin connects separate LEC islands. (D) At day 4 on Collagen 1 + fibronectin, LECs showed tightened junctions and fibronectin was localized in the junctional area.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Expressing

Journal: Microcirculation (New York, N.Y. : 1994)

Article Title: A bioengineered lymphatic vessel model for studying lymphatic endothelial cell-cell junction and barrier function

doi: 10.1111/micc.12730

Figure Lengend Snippet: (A) Activated integrin α5 was visualized in both ECM composition conditions by using anti-integrin α5 antibody (clone: SNAKA51) that can only detect the activated form of the integrin α5. F-actin was also observed in these conditions. (B) LECs in Collagen 1 were pre-treated with anti-integrin α5 antibodies (clone: SNAKA51) antibodies to activate integrin α5 in LECs. The fixed samples were stained with anti-VE-cadherin antibodies, anti-JAM-A antibodies, and phalloidin to visualize adherens junctions and F-actin. (C) Quantification of the relative junction area was performed, illustrating a significantly higher junction area in integrin α5 activated cells compared to the control LECs. ** p = 0.0020; Two tailed unpaired Student t-test, n = 6 per group. Data are expressed as mean ± S.E.M. (D) Control LECs or LECs with activated integrin α5 were seeded in LV-on-chip and cultured for 3 days on the rocking platform. 70 kDa dextran was introduced to the lymphatic lumens. Dextran diffusion was observed at 0 and 1 minutes under microscopy. Superimposed red dashed lines represent the edges of the vessel lumens. (E) Quantification of the permeability of LEC-generated engineered LVs in collagen 1 with and without integrin α5 activation. ** p = 0.0021. Two tailed unpaired Student t-test, n = 5 per group. Data are expressed as mean ± S.E.M. (F) This table summarizes our findings regarding LEC permeability and integrin α5 activity. LVs grown in Collagen 1 without any activator treatment showed high LEC permeability and low integrin α5 activity. In contrast, LVs grown in either Collagen 1 + Fibronectin or LVs grown in only Collagen 1 with integrin α5 activator pre-treatment both showed low LEC permeability and high integrin α5 activity.

Article Snippet: In the hollow channel, we seeded human dermal microvascular lymphatic endothelial cells (LECs) to form a biomimetic lymphatic vessel ( ).

Techniques: Staining, Control, Two Tailed Test, Cell Culture, Diffusion-based Assay, Microscopy, Permeability, Generated, Activation Assay, Activity Assay

Journal: The Journal of Biological Chemistry

Article Title: Human Gb3/CD77 synthase produces P1 glycotope-capped N-glycans, which mediate Shiga toxin 1 but not Shiga toxin 2 cell entry

doi: 10.1016/j.jbc.2021.100299

Figure Lengend Snippet: Quantitative analysis of A4GALT transcripts in CHO-Lec2 cells

Article Snippet: CHO-Lec2 cells were obtained from the American Type Culture Collection.

Techniques:

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Representative immunofluorescence images of AIDS-KS lesion tissues (lower) and their adjacent normal skin tissues (upper) stained with antibodies against mesenchymal (Nestin, PDGFRA, or α-SAM in green), endothelial (PDPN, CD31 or VEGFR2 in red), and KSHV (LANA in yellow) markers. The nuclei were counterstained with Hoechst 33342 (blue). Scale bars, 50 μm. Images of each individual channel for mesenchymal, endothelial and KSHV marker, respectively, are shown in . ( B) Triple labeling for mesenchymal, endothelial, and KSHV markers, demonstrating the colocalization of these three fluorescent signals in the same KS tumor cell, as revealed by the plot of fluorescence intensity profiles across a white arrow in panel A. (C) Co-expression of mesenchymal (Nestin, PDGFRA, and α-SAM), endothelial (PDPN, CD31, and VEGFR2), and KSHV (LANA) markers in KS early (patch and plaque) and late lesions (nodular). Boxed areas are enlarged. Scale bar, 50 μm. ( D) Number of LANA, Nestin, CD31, PDGFRA, PDPN, α-SAM, and VEGFR2 positive cells was counted from 4–6 individual fields (180μm x 150μm) composed mostly of spindle tumor cells and vessels in KS early (n = 7 samples) or late lesions (n = 5 samples). Numbers were compared by Chi-2 test. ( E) Spearman’s test shows a correlation between LANA expression and cells positive for PDPN in early and late KS tumors. ( F) Percentage of Nestin-/CD31-, Nestin+/CD31-, Nestin+/CD31+, Nestin-/CD31+ and PDGFRA-/PDPN-, PDGFRA+/PDPN-, PDGFRA+/PDPN+, PDGFRA-/PDPN+, and α-SAM-/VEGFR2-, α-SAM+/VEGFR2-, α-SAM+/VEGFR2+, α-SAM-/VEGFR2+ cells in LANA+ spindle tumor cells in early or late KS lesions.

Article Snippet: Human dermal lymphatic endothelial cells (LECs, ScienCell) were cultured in endothelial cell medium (ECM, ScienCell) plus supplements (ECGS, ScienCell).

Techniques: Immunofluorescence, Staining, Marker, Labeling, Fluorescence, Expressing

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) The difference in morphology and structure between normal vessels (left) and KS specialized vessels (right). Higher magnifications of the black-boxed areas are shown underneath. Scale bar, 200 μm (upper), 50 μm (lower). Asterisk, KS abnormal vessels. (B) Expression patterns of mesenchymal and endothelial markers in normal and KS abnormal vessels. Samples were stained with antibodies against Nestin/PDGFRA/α-SAM (green), CD31/PDPN /VEGFR2 (red), and LANA (yellow), and the nuclei were counterstained with Hoechst 33342 (blue). Scale bar, 50 μm. Asterisk, KS abnormal vessels. (C) LANA+ abnormal hybrid vascular density in early and late KS tumors. The number of vessels was quantified from 4–6 individual fields (354μm x 246μm) for each KS tumor sample. Error bars represent mean ± SEM. All statistical analyses were performed using the Mann-Whitney U test. *p < 0.05, **p < 0.01, ***p < 0.001, NS, not significant.

Article Snippet: Human dermal lymphatic endothelial cells (LECs, ScienCell) were cultured in endothelial cell medium (ECM, ScienCell) plus supplements (ECGS, ScienCell).

Techniques: Expressing, Staining, MANN-WHITNEY

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Cell lysates from mock- and KSHV-infected PDLSCs at indicated time points were immunoblotted for PDGFRA, COL1A1, α-SAM, TAGLN, PROX1, PDPN, VEGFA, and β-actin. ( B) Relative mRNA levels of mesenchymal and endothelial related genes in mock- and KSHV-infected PDLSCs (K-PDLSCs) after 4 days infection. ( C) Mock- and KSHV-infected PDLSCs (2-D) were immunostained for LANA, TAGLN, PDPN, vWF, and CD31 at 4 days post-infection. Scale bar, 50 μm. ( D) A time course of K-PDLSC aggregating to form spheroid in non-adherent plates. Scale bar, 500 μm. ( E) Expression of LANA, TAGLN, PDPN, vWF, and CD31 in mock- and KSHV-infected PDLSC spheroid (3-D) at 4 days post-infection. Scale bar, 50 μm. ( F) The expression of endothelial markers in mock- and KSHV-infected PDLSCs under 2-D or 3D cell culture for 4d. ( G) The mRNA expression level of TAGLN , α-SAM , Nestin , PDGFRA , PDPN , ICAM , PROX1 , CD31 , and VEGFA was analyzed by RT- qPCR in K-PDLSC spheroids in comparison with their parallel 2D culture.

Article Snippet: Human dermal lymphatic endothelial cells (LECs, ScienCell) were cultured in endothelial cell medium (ECM, ScienCell) plus supplements (ECGS, ScienCell).

Techniques: Infection, Expressing, Cell Culture, Quantitative RT-PCR, Comparison

Journal: PLoS Pathogens

Article Title: Kaposi’s sarcoma-associated herpesvirus vFLIP promotes MEndT to generate hybrid M/E state for tumorigenesis

doi: 10.1371/journal.ppat.1009600

Figure Lengend Snippet: (A) Mock- and KSHV-infected PDLSCs and LECs were examined for PDGFRA and PDPN expression profile by flow cytometry analysis. Three subpopulations (xM, hybrid M/E, and xE) were quantified based on the PDGFRA and PDPN profiles ( n = 3 independent experiments). Statistical analyses were performed using two-tailed Student’s test and P-values were calculated by GraphPad Prism. *p < 0.05, **p < 0.01, ***p < 0.001. ( B) PDGFRA and PDPN expression in PDLSC and K-PDLSC spheroids at 4 days post-infection were analyzed by IFA. Scale bar, 50 μm. ( C) K-PDLSCs were stained for PDGFRA and PDPN and sorted by flow cytometry. The purified xM and M/E populations were cultured for 10 days and their PDGFRA/PDPN profiles were examined for their phenotypic plasticity. ( D) Western blot analysis of xM, M/E, and LECs for their mesenchymal and endothelial markers. ( E) The expression profiles of mesenchymal and endothelial markers in hybrid M/E and xM state cells were analyzed at the mRNA level by RT-qPCR. ( F) IFA analysis of LANA, TAGLN, and VCAM in xM and M/E state cells. Scale bar, 50 μm.

Article Snippet: Human dermal lymphatic endothelial cells (LECs, ScienCell) were cultured in endothelial cell medium (ECM, ScienCell) plus supplements (ECGS, ScienCell).

Techniques: Infection, Expressing, Flow Cytometry, Two Tailed Test, Staining, Purification, Cell Culture, Western Blot, Quantitative RT-PCR