primary human glomerular microvascular endothelial cells  (PromoCell)

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Modular Microphysiological System for Modeling of Biologic Barrier Function

doi: 10.3389/fbioe.2020.581163

Figure Lengend Snippet: Recapitulating alveolar microenvironment: (A) Cartoon of alveolar expansion during inspiration. Created with BioRender.com. (B) Calculated bi-axial strain in response to applied pressure in the microfluidic chip. (C) Application of a cyclic pressure in the apical channel using a sine wave to mimic breathing at approximately 20 breaths per minute. A constant pressure was applied to the basal channel to drive the flow of PBS. (D) Workflow for development of ALI co-culture. Endothelial cells (LMECs) were first seeded on the basal surface of a transwell culture insert, followed alveolar epithelial cells (AECs). Dexamethasone was added to the apical chamber on Day 3 to enhance the epithelial cell barrier. On Day 7, the ALI was induced by removing media from the apical chamber. On Day 10, the membrane was cut from the transwell support and bonded into the microfluidic chip and exposed to dynamic strain. (E) TEER measurements during liquid-liquid co-culture in the transwell with (Dex +) and without (Dex-) dexamethasone. Statistical analysis by unpaired t -test ( N = 3). (F) 3D visualization of fluorescent z -stack after culture for 24 h under cyclic pressure exposure in the MPS. Cytokeratin (green) staining shows the alveolar endothelial cells on the apical side of the membrane, while PECAM1 (red) staining indicates the lung microvascular endothelial cells on the basal side.

Article Snippet: Primary human glomerular microvascular endothelial cells (GMECs; Cell Systems) were culture per the manufacturer in complete endothelial cell growth media 2 (PromoCell).

Techniques: Co-Culture Assay, Membrane, Staining

Journal: Frontiers in Bioengineering and Biotechnology

Article Title: Modular Microphysiological System for Modeling of Biologic Barrier Function

doi: 10.3389/fbioe.2020.581163

Figure Lengend Snippet: Modeling glomerular filtration barrier; (A) Cartoon of how glomerular anatomy was modeled within the MPS (B) Schematic representation of the workflow for the development of the co-culture model of the GFB. (C) TEER measurements of podocytes only (Green), endothelial cells only (Red), and co-culture (black) during static culture on a transwell insert over the course of 5 days. Data presented as mean ± standard deviation. Statistical analysis by unpaired t-test ( N = 3; * indicates Co-Culture vs. CiPodos; # indicates GMECs vs. CiPodos). * p < 0.05, # p < 0.05. (D) Urinary filtration of albumin in 1 h on the microfluidic device. The significant decrease in the urinary filtration of albumin between the co-culture, despite similar TEER values, indicates a functional barrier as opposed to a physical barrier. Statistical analysis by one-way ANOVA with Tukey’s multiple comparisons ( N = 5; * indicates Co-Culture vs. CiPodos; # indicates GMECs vs. CiPodos). *** p < 0.0001, ### p < 0.0001. (E) Tight junctions observed in endothelial cells following culture on the MPS (F) Presence of nephrin in CiPodos after culture in the MPS.

Article Snippet: Primary human glomerular microvascular endothelial cells (GMECs; Cell Systems) were culture per the manufacturer in complete endothelial cell growth media 2 (PromoCell).

Techniques: Filtration, Co-Culture Assay, Standard Deviation, Functional Assay

Journal: The Journal of Biological Chemistry

Article Title: CLIC5A binds to and stabilizes the open and active conformation of ezrin

doi: 10.1016/j.jbc.2025.110646

Figure Lengend Snippet: ERM and Rac-1 activation are amplified by the CLIC5A/ezrin interaction. A , endogenous pERM abundance in lysates and detergent resistant pellets of COS-7 cells transfected with GFP-CLIC5A cDNA and increasing concentrations of GFP-ezrin 432-586 (T567D) cDNA . GFP-CLIC5A cDNA was kept constant and the GFP-CLIC5A: GFP-ezrin 432-586 (T567D) cDNA transfection ratio was 1:1, 1:2, 1:4, and 1:8. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± SD, n = 3 independent experiments, one-way ANOVA: F = 7.90; p = 0.0007. p values shown represent post hoc Dunnett’s multiple comparisons). B , pERM abundance in lysates and detergent resistant pellets of COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP- e zrin 432-570 cDNA. Left panel , representative WB. Right panel, densitometric quantification of the endogenous pERM: endogenous ezrin ratio (mean ± S.D., n = 3 biologically independent experiments, two-way ANOVA: interaction F = 12.25; p = 0.01; CLIC5A effect: F = 39.63, p = 0.0002; ezrin 432-570 effect F = 22.58. p = 0.0014; p values shown represent post hoc Tukey’s multiple comparisons). C , endogenous Rac1 WB for COS-7 cell lysates and endogenous Rac1-GTP captured by PAK-PBD pulldown (PD) from COS-7 cells transiently transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of GFP-ezrin 432-586 (T567D) cDNA. Left panel, representative WB. Right panel: densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± SD, n = 3 independent experiments, two-way ANOVA: interaction F = 6.32, p = 0.036; CLIC5A effect F = 29.25, p = 0.001; ezrin 432–586 effect F = 5.88; p = 0.042, p values for post hoc Tukey’s multiple comparisons are shown). D , PAK-PBD pulldown (PD) of endogenous Rac1-GTP from lysates of COS-7 cells transfected with GFP-CLIC5A cDNA with or without an 8-fold excess of transiently expressed GFP-ezrin 432 - 570 cDNA. Left panel, representative WB. Right panel, densitometric quantification of endogenous Rac1-GTP/total endogenous Rac1 (mean ± S.D., n = 3 independent experiments, two-way ANOVA: interaction F = 0.029, p = 0.87; CLIC5A effect F = 62.17 p < 0.0001; ezrin 432–570 effect F = 0.012; p = 0.91, p values for post hoc Tukey’s multiple comparisons are shown). E , endogenous Rac1-GTP abundance in human glomerular endothelial cells (hGENs) determined by Rac1-GTP G-LISA. The cells were transduced with control adenoviral-vector (ad-Vector) or untagged CLIC5A cDNA in the same vector (ad- CLIC5A ) at an increasing multiplicity of infection (MOI) (mean ± SD, n = 3 independent experiments). F , representative WB of lysates from hGEN cells transduced with ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA. G , change relative to baseline of Rac1-GTP in hGEN cells transduced with 30 MOI ad-Vector or ad- CLIC5A with or without ezrin-specific siRNA (mean ± SD, n = 4 independent experiments, two-way ANOVA: interaction F = 6.35, p = 0.027; CLIC5A effect F = 34.18, p < 0.0001; ezrin siRNA effect F = 6.86; p = 0.023, p values shown represent post hoc Tukey’s multiple comparisons). H , coimmunoprecipitation of endogenous Rho-GDI with endogenous ezrin in the presence and absence of transiently expressed GFP-CLIC5A. Left panel: representative α-Rho GDI, α-ezrin, and α-CLIC5A WB of lysates (input) and α-ezrin immunoprecipitates (IP). Right panel: Quantification of endogenous Rho-GDI and endogenous ezrin immunoprecipitated with α-ezrin antibodies (n = 3 biologically distinct experiments, mean ± SD, Student’s t test). CLIC, chloride intracellular channel; PBD, protein binding domain; pERM, phosphorylated ezrin, radixin, and moesin proteins; Rho-GDI, Rho guanine nucleotide dissociation inhibitor; WB, Western blot.

Article Snippet: Mycoplasma-free primary human glomerular endothelial cells (hGENs) were purchased from Angio-Proteomie (# cAP-0004) and maintained in EGM-2 MV Bulletkit growth media (# CC-3162, Lonza) containing 5% FBS and 1% penicillin/streptomycin at 37 °C in humidified air containing 5% CO 2 .

Techniques: Activation Assay, Amplification, Transfection, Transduction, Control, Plasmid Preparation, Infection, Immunoprecipitation, Protein Binding, Western Blot