Journal: PLoS ONE

Article Title: Delta/Notch-Like EGF-Related Receptor (DNER) Is Not a Notch Ligand

doi: 10.1371/journal.pone.0161157

Figure Lengend Snippet: (A) Pooled luciferase results from 4 separate experiments (normalized to the mean of empty vector in each experiment). U2OS cells were transfected with ligand (DLL1, DNER, or EV), and separately a population of U2OS cells was transfected to express Notch, the control luciferase Renilla , and TP1, a promoter that expresses firefly luciferase when Notch is activated. The two populations were co-cultured 24 hours after transfection ( trans configuration), and activity read after an additional 24–48 hours of incubation. (B) C2C12 cells (myoblasts) were incubated with differentiation media (2% horse serum) that either had pre-clustered DLL1-fc (1:1), pre-clustered DNER-fc (1:1), un-clustered DNER-fc, or fc only, all at a ratio of 1:150 in media. Cells were incubated for 72 hours, then fixed, and stained for the presence of myosin heavy chain (MHC) and nuclei. By measuring the percent of total nuclei that were inside of differentiated MHC positive myotubes, fusion indexes were calculated. (C) DNER (top left, green) transfected U2OS cells were not labeled by pre-clustered Notch-fc (top middle, red) but DLL1 (bottom left, green) transfected U2OS cells were labeled by pre-clustered Notch-fc (bottom middle, red). Merged images are shown at far right. Scale 10 μM. **** = p value <0.0001. *** = p value 0.002. ns = not significant. DLL1 = Delta-like 1, a known Notch Ligand, DNER = Delta/Notch-like epidermal growth factor (EGF) related receptor, GSI = γ-secretase inhibitor, fc only = rabbit anti-human-fc.

Article Snippet: Transient transfection of cultured cells was accomplished with the Fugene/Optimem (Promega) system and according to the manufacturer’s instructions with the following plasmids at a concentration, unless otherwise stated, of 0.1 μg/well of DNA per plasmid of a 96-well plate: DNER (generous gift of Drs. De Graaff and Sillevis-Smitt, Erasmus Medical Center, Rotterdam the Netherlands, originally from M. Kengaku), DLL1 (EX-Y3540-M11, GeneCopoeia, Rockville, MD, USA), empty vector on a p-receiver backbone (GeneCopoeia, Rockville, MD, USA), Renilla (luciferase control, 0.01 μg/well, pRL-TK, Promega E2241), TP1 (very sensitive intracellular Notch reporter with twelve CSL binding sites converting intracellular Notch activity to firefly luminescence, which is capable of detecting slight perturbations in signaling strength with little background), and full length human Notch1 on a MigR1 backbone (both generous gifts of Dr. Jon Aster).

Techniques: Luciferase, Plasmid Preparation, Transfection, Control, Cell Culture, Activity Assay, Incubation, Staining, Labeling

Journal: Biochemical and biophysical research communications

Article Title: CALCINEURIN INHIBITORS AUGMENT ENDOTHELIAL-TO-MESENCHYMAL TRANSITION BY ENHANCING PROLIFERATION IN ASSOCIATION WITH CYTOKINE-MEDIATED ACTIVATION

doi: 10.1016/j.bbrc.2019.09.043

Figure Lengend Snippet: A) EC (5 ×103 cells per well) were cultured in the absence or presence of FK506 or CsA, and proliferation was evaluated by 3[H]-Thymidine incorporation after 72 hours. Treatment with VEGF-A served as a positive control. Bar graphs represent the fold change in proliferation (mean cpm± SEM) of 12 experiments run in triplicate (*P<0.05; **P<0.01). B) EC were aggregated into spheroids and embedded in collagen matrix in the absence/presence of FK506, CsA or VEGF-A as described in Methods. Photomicrographs are representative of ~ 5 spheroids per condition in n=3 independent experiments. C) EC were cultured with FK506 or CsA in the absence/presence of anti-VEGF-A. Bar graphs represent the fold change in proliferation (mean cpm ± SEM of n≥3 experiments). *P<0.05 and **P<0.01 (within each group). D) EC were cultured with FK506 or CsA for 30 mins and pY1175VEGFR2 and total VEGFR2 expression was evaluated by Western blot. Numbers below each blot represent the densitometric analysis of the ratio of phoshoprotein to loading control (of n≥3 experiments). E) CHO-AA8 or CHO-VEGF cells (1 × 105) were injected subcutaneously into the ears of nude mice that received FK506 (1 mg/kg) or CsA (3 mg/kg) by daily i.p. injection. Photographs were taken on day 4. F) Immunohistochemistry using anti-CD31 of day 4 injected ears from Panel E. The images in panels E and F are representative of n=3 mice.

Article Snippet: AA8 cells (Clontech Laboratories, Palo Alto, CA) were used as negative controls.

Techniques: Cell Culture, Positive Control, Expressing, Western Blot, Injection, Immunohistochemistry

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of ECM on the differentiation of kidney organoids in a static culture condition. A Fluorescent images of immunostained kidney organoids cultured with various conditions of ECMs for 6 days. The cells were immunostained by NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue). B Quantitative analysis of kidney antibody-specific markers from kidney organoids (* indicates p <0.05 for experimental group vs. control group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Cell Culture

Journal: Nano Convergence

Article Title: Effect of biochemical and biomechanical factors on vascularization of kidney organoid-on-a-chip

doi: 10.1186/s40580-021-00285-4

Figure Lengend Snippet: Effect of the shear stress on the differentiation of kidney organoids in a kidney organoid-on-a-chip. A Fluorescent images showing hPSCs-derived kidney organoid-related cell differentiation under a shear stress condition. Kidney organoids were cultured within kidney organoid-on-a-chip for 6 days. After culturing for 6 days, the cells were stained for NPHS1 (podocyte, red), PECAM1 (vascular endothelial cell, green), LTL (proximal tubules, white), and DAPI (nuclei, blue) to identify the kidney-related cells, respectively. B Quantitative analysis of the kidney-related markers from kidney organoids. (* indicates p < 0.05 for LTL expression of Matrigel-coated group vs. Matrigel/VEGF-coated group, *** indicates p < 0.001 for PECAM expression of Matrigel-coated group vs. Matrigel/VEGF-coated group). Scale bars are 100 μm

Article Snippet: Primary antibodies against the following proteins were used to characterize various kidney-related cell types: podocytes (anti-NPHS1, R&D AF4269, 1:500), proximal tubular cells (anti-LTL, Vector Labs FL‐1321, 1:500), and ECs (anti-PECAM1, Abcam ab9498, 1:200).

Techniques: Derivative Assay, Cell Differentiation, Cell Culture, Staining, Expressing