Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces GSK-3beta activation in dose- and time-dependent manners in HPMECs. Expression of P-GSK-3beta and GSK-3beta was detected after incubation with different concentrations of LPS for 1 h (A) . The expression of P-GSK-3beta was represented as a histogram according to band intensities (B) . Expression of P-GSK-3beta and GSK-3beta was examined at indicated time points after stimulation with LPS (0.1 μg/ml) in HPMECs (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Expressing, Incubation, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: Involvement of GSK-3beta in LPS-induced GEF-H1/ROCK signaling activation. HPMECs were incubated with LPS (0.1 μg/ml) at different indicated times, and the GEF-H1 and myosin-associated phosphatase type 1 (P-MYPT 1: the substrate of ROCK) were detected by Western blot assay (A) . The expression of GEF-H1 and P-MYPT 1 were represented as a histogram according to band intensities (B) . * < 0.05 vs. LPS un-treatment group. Inhibition effect of GSK-3beta activity in HPMECs was analyzed by Western blot (C,D) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group. HPMECs were pretreated with SB-216763 (20 μM) for 1 h and then were exposed to LPS (0.1 μg/ml) for 1 h. The expression of GEF-H1 and P-MYPT 1 were determined by Western blot (E) . The Western blotting results are presented as a histogram showing the band intensity values (F) . * P < 0.05 vs. the negative control group, # P < 0.05 vs. the corresponding LPS treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Activation Assay, Incubation, Western Blot, Expressing, Inhibition, Activity Assay, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta signaling is involved in LPS-induced HPMECs barrier disruption. The HPMECs were plated on the gold microelectrodes. When HPMECs formed monolayers and reached stable TER values, the SB-216763 (20 μM) was added. After 1 h, the medium or LPS (0.1 μg/ml) was added for another 6 h. The HPMEC monolayers permeability was determined by real-time TER measurement (A) . The results of the 3 h LPS stimulation were represented as a histogram in (B) according to the TER curves. * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Permeability, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: LPS induces degradation of beta-catenin and ZO-1 in HPMECs monolayer. LPS (0.1 μg/ml) induced down-regulation of ZO-1 expression and increase of phosphorylated degradation of beta-catenin in a time-dependent manner (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . * P < 0.05 vs. LPS un-treatment group.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Expressing, Western Blot

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK signaling is required for LPS-induced degradation of beta-catenin and ZO-1. After transfection with GEF-H1 siRNA and Control siRNA for 48 h, HPMECs were treated with SB-216763 (20 μM) and/or Y-27632 (10 μM) for another 1 h prior to LPS stimulation (0.1 μg/ml) for 3 h. The expression of ZO-1 was determined by immunoblotting, and GAPDH protein was used as loading control (A) . The Western blotting results are presented as a histogram showing the band intensity values (B) . The expression of P-beta-catenin was determined by immunoblotting, and GSK-3beta and GAPDH proteins were used as control (C) . The Western blotting results are presented as a histogram showing the band intensity values (D) . * P < 0.05 vs. negative control. # P < 0.05 vs. corresponding LPS-stimulated group. NS, no significance.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Transfection, Control, Expressing, Western Blot, Negative Control

Journal: Frontiers in Cellular and Infection Microbiology

Article Title: GSK-3Beta-Dependent Activation of GEF-H1/ROCK Signaling Promotes LPS-Induced Lung Vascular Endothelial Barrier Dysfunction and Acute Lung Injury

doi: 10.3389/fcimb.2017.00357

Figure Lengend Snippet: GSK-3beta/GEF-H1/ROCK pathway is involved in LPS-induced HPMECs barrier disruption by beta-catenin and ZO-1. HPMECs monolayer was pretreated with SB-216763 (20 μM) (A) , GEF-H1 siRNA (B) , or Y-27632 (10 μM) (C) , for indicated times and then was exposed to LPS (0.1 μg/ml) for 3 h before fixation and staining with anti-beta-catenin and anti-ZO-1 antibody as described in Materials and Methods. Beta-catenin (green) and ZO-1 (green) were visualized by immunofluorescence microscopy. Red arrows not only represent the expression of beta-catenin and ZO-1 in the membrane of HPMECs but also represent the cell-cell gaps formation in the ECs monolayer.

Article Snippet: Primary human pulmonary micro-vascular endothelial cells (HPMECs) were obtained ScienCell Research Laboratories and maintained in ScienCell Endothelial Cell Medium in a humidified 37°C, 5% CO 2 incubator.

Techniques: Disruption, Staining, Immunofluorescence, Microscopy, Expressing, Membrane

Journal: Acta biomaterialia

Article Title: Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform

doi: 10.1016/j.actbio.2020.02.015

Figure Lengend Snippet: A summary of each organoid or tissue construct type with the cells making them up. Also described are the various cell sources and cell types (primary, cell line, iPSC-derived, etc.), relative percentage in each model, and documented tissue specific functionalities.

Article Snippet: Organoid or tissue construct type Cell types cell source Type of cell Percentage in model Documented functionality Citation Liver Hepatocytes Bioreclamation Primary human 75% Albumin and urea secretion; Drug metabolism; Toxicity responses [ 18 , 47 ] Stellate cells Bioreclamation Primary human 10% Kupffer cells Sekisui XenoTech Primary human 10% Liver-Derived endothelial cells Lonza primary human 5% Cardiac Cardiomyocytes Ncardia iPSC 75% Spontaneous beating; Beating changes in response to drugs; Toxicity responses [ 18 , 47 ] Cardiac fibroblasts ScienCell Primary HUMAN 20% Cardiac endothelial cells ScienCell Primary human 5% Lung (3-tissue system) A549 Alveolar epithelial cells Lonza Human cell line 100% n/a – Lung (6-tissue system) Lung fibroblasts Lonza Primary human 20% n/a – Human bronchial epithelial cells Lonza Primary human 80% Endothelial HUVEC Lonza Primary human 100% n/a – Testis Spermatogonial stem cells Donor via NDRI Primary human ~80% Androgen production; Supporting germ cell differentiation; Toxicity responses; Virus replication [ 34 – 36 ] Leydig cells Donor via NDRI Primary human ~10% Sertoli cells Donor via NDRI Primary human ~10% Peritubular cells Donor via NDRI Primary human Contaminant of SSC populations Brain Brain microvascular endothelial cells Cell Systems Primary human 30% Barrier function to the middle of the spheroid [ 37 ] Brain vascular pericytes ScienCell Primary human 15% Astrocytes ScienCell Primary human 15% Microglia Tempo Bioscience IPSC 5% Oligodendrocytes Tempo Bioscience Oligodendrocyte progenitor cells 15% Neural cells Axol Biosciences Neural stem cells 20% Open in a separate window A summary of each organoid or tissue construct type with the cells making them up.

Techniques: Construct, Cell Differentiation, Virus

Journal: Acta biomaterialia

Article Title: Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform

doi: 10.1016/j.actbio.2020.02.015

Figure Lengend Snippet: Drug toxicity assessment of ifosfamide in a 6-organoid system. L/D imaging of liver, cardiac, lung, endothelial, brain, and testis organoids under Control (no drug, a–c), Condition 1 (with ifosfamide and liver organoid, d–f), and Condition 2 (with ifosfamide and without liver organoid, g and h). In Condition 1, the metabolized drug caused downstream toxicity in the brain organoid; in Condition 2, no significant toxicity occurred. Green stain: calcein AM-stained viable cells; Red stain: ethidium homodimer 1-stained dead cells. Scale bar represents 100 μM. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Organoid or tissue construct type Cell types cell source Type of cell Percentage in model Documented functionality Citation Liver Hepatocytes Bioreclamation Primary human 75% Albumin and urea secretion; Drug metabolism; Toxicity responses [ 18 , 47 ] Stellate cells Bioreclamation Primary human 10% Kupffer cells Sekisui XenoTech Primary human 10% Liver-Derived endothelial cells Lonza primary human 5% Cardiac Cardiomyocytes Ncardia iPSC 75% Spontaneous beating; Beating changes in response to drugs; Toxicity responses [ 18 , 47 ] Cardiac fibroblasts ScienCell Primary HUMAN 20% Cardiac endothelial cells ScienCell Primary human 5% Lung (3-tissue system) A549 Alveolar epithelial cells Lonza Human cell line 100% n/a – Lung (6-tissue system) Lung fibroblasts Lonza Primary human 20% n/a – Human bronchial epithelial cells Lonza Primary human 80% Endothelial HUVEC Lonza Primary human 100% n/a – Testis Spermatogonial stem cells Donor via NDRI Primary human ~80% Androgen production; Supporting germ cell differentiation; Toxicity responses; Virus replication [ 34 – 36 ] Leydig cells Donor via NDRI Primary human ~10% Sertoli cells Donor via NDRI Primary human ~10% Peritubular cells Donor via NDRI Primary human Contaminant of SSC populations Brain Brain microvascular endothelial cells Cell Systems Primary human 30% Barrier function to the middle of the spheroid [ 37 ] Brain vascular pericytes ScienCell Primary human 15% Astrocytes ScienCell Primary human 15% Microglia Tempo Bioscience IPSC 5% Oligodendrocytes Tempo Bioscience Oligodendrocyte progenitor cells 15% Neural cells Axol Biosciences Neural stem cells 20% Open in a separate window A summary of each organoid or tissue construct type with the cells making them up.

Techniques: Imaging, Staining

Journal: Acta biomaterialia

Article Title: Probing prodrug metabolism and reciprocal toxicity with an integrated and humanized multi-tissue organ-on-a-chip platform

doi: 10.1016/j.actbio.2020.02.015

Figure Lengend Snippet: Viability quantification under insult by ifosfamide in a 6-organoid system. Quantification of live cell ratios (L/L + D) for liver, cardiac, lung, endothelial, brain, and testis organoids under Control (no drug), Condition 1 (drug with liver organoid), and Condition 2 (drug without liver organoid). A significant reduction in viability is observed for the brain organoid under Condition 1 (red). Statistical significance: ** p < 0.05. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: Organoid or tissue construct type Cell types cell source Type of cell Percentage in model Documented functionality Citation Liver Hepatocytes Bioreclamation Primary human 75% Albumin and urea secretion; Drug metabolism; Toxicity responses [ 18 , 47 ] Stellate cells Bioreclamation Primary human 10% Kupffer cells Sekisui XenoTech Primary human 10% Liver-Derived endothelial cells Lonza primary human 5% Cardiac Cardiomyocytes Ncardia iPSC 75% Spontaneous beating; Beating changes in response to drugs; Toxicity responses [ 18 , 47 ] Cardiac fibroblasts ScienCell Primary HUMAN 20% Cardiac endothelial cells ScienCell Primary human 5% Lung (3-tissue system) A549 Alveolar epithelial cells Lonza Human cell line 100% n/a – Lung (6-tissue system) Lung fibroblasts Lonza Primary human 20% n/a – Human bronchial epithelial cells Lonza Primary human 80% Endothelial HUVEC Lonza Primary human 100% n/a – Testis Spermatogonial stem cells Donor via NDRI Primary human ~80% Androgen production; Supporting germ cell differentiation; Toxicity responses; Virus replication [ 34 – 36 ] Leydig cells Donor via NDRI Primary human ~10% Sertoli cells Donor via NDRI Primary human ~10% Peritubular cells Donor via NDRI Primary human Contaminant of SSC populations Brain Brain microvascular endothelial cells Cell Systems Primary human 30% Barrier function to the middle of the spheroid [ 37 ] Brain vascular pericytes ScienCell Primary human 15% Astrocytes ScienCell Primary human 15% Microglia Tempo Bioscience IPSC 5% Oligodendrocytes Tempo Bioscience Oligodendrocyte progenitor cells 15% Neural cells Axol Biosciences Neural stem cells 20% Open in a separate window A summary of each organoid or tissue construct type with the cells making them up.

Techniques:

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a-d , Ifnar-/- mice were infected with ZIKV as above, and the choroid plexuses were isolated at 4 DPI. a , Choroid plexuses were stained with antibodies against TTR (choroid plexus epithelial cell maker, green) and ZIKV-E protein (red). left : three layers of confocal images (labeled with Top, Middle, and Bottom) along the Z-axis. right : a three-dimensional reconstruction of the z-stacked image, highlighting the ZIKV-infected cells in the stroma layer of the choroid plexus. The top, middle, and bottom layers showed TTR+/ZIKV-, TTR-/ZIKV+, and TTR+/ZIKV-, respectively. A representative image is shown. b , Choroid plexuses were costained with FITC-IB4 for capillaries (green), anti-PDGFβR antibody for pericytes (purple), and anti-ZIKV-E antibody for the Zika envelope protein (red). Nuclei were stained with Hoechst 3342 stain (blue). The ZIKV-E signal colocalized with PDGFβR. For a and b , scale bars = 20 μm. c-d , ChP (n = 6, two from three mice) were harvested following cardiac perfusion and stained with anti-PDGFβR antibody for pericytes, anti-ZIKV-E antibody for the Zika envelope protein, and Hoechst 33342 for nuclei. (See for detail). The percentage of ZIKV-infected cells in the total ChP stromal cells(c) and PDGFβR (+) populations in the ZIKV (+) cells (d) were enumerated by using Imaris image analysis software. Each symbol represents an individual choroid plexus** P <0.01 (Student’s t-test). e , An electron micrograph showing a pericyte infected with ZIKV on a choroid plexus capillary. Pseudocolors (green and blue) indicate the cytoplasm and the nucleus, respectively, of a choroid plexus endothelial cell (EC) facing the lumen of a capillary (LUMEN). Patches of electron-dense particles (arrowheads) were found in a pericyte next to the EC. f , A higher magnification of the image from e shows that the particles have characteristics typical of flavivirus nucleocapsids. fi , A higher magnification image of the inset area of Fig e , Scale bars: 800 nm in e and 100 nm in f and fi .

Article Snippet: Primary human brain vascular pericytes (HBVPs) were obtained from ScienCell (Cat No. 1200) and maintained using the manufacturer’s protocol and their pericyte culture media with 2% FBS, 1 x pericyte growth supplement, and 1 x penicillin and streptomycin.

Techniques: Infection, Isolation, Staining, Labeling, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a , ZIKV replication in primary pericytes from mouse choroid plexus (IFNAR +/- ). Cells (three independent wells) were infected with ZIKV, PLCal_ZV (MOI = 0.1) and grown in the presence of 5 μg/mL mouse anti-IFNAR-1 neutralizing antibody (clone MAR 5A3, ɑ-IFNAR) or isotype control antibody (clone MOPC-21). The cell culture supernatant was harvested at 5 DPI, and the virus titer was enumerated. * P <0.05 (Student’s t-test) b , Primary human brain vascular pericytes (five independent wells) were infected with ZIKV as in a and then incubated in the presence/absence of a human type 1 IFN neutralizing antibody mixture (ɑ-IFN Aby). The supernatants were harvested every 24 hours for five days, and the virus titers were enumerated with a virus titration assay. ** P < 0.005 (two-way ANOVA). c , Primary human brain vascular pericytes were infected with ZIKV and ZIKV-infected cells were enumerated by using FACS with anti ZIKV-E (clone 4G2). Each dot represents cells from single well. d , Mock- or ZIKV-infected HBVP were stained with antibodies against PDGFβR (red) and ZIKV-E protein (green). Two consecutive confocal layers were projected into a single layer with the Z-project function of ImageJ software (version 2.0.0). e , HBVP infected with ZIKV were stained with antibodies against AXL (green), PDGFβR (magenta) and ZIKV-E protein (red). Scale bars = 20 μm. f and g , HBVP were pre-treated with antibody for three hours then infected with ZIKV (m.o.i. = 1). Three days later, viral RNA ( f ) and progeny virus titers in the supernatants ( g ) were analyzed. **** P < 0.001 (one-way ANOVA).

Article Snippet: Primary human brain vascular pericytes (HBVPs) were obtained from ScienCell (Cat No. 1200) and maintained using the manufacturer’s protocol and their pericyte culture media with 2% FBS, 1 x pericyte growth supplement, and 1 x penicillin and streptomycin.

Techniques: Infection, Control, Cell Culture, Virus, Incubation, Titration, Staining, Software

Journal: PLoS Pathogens

Article Title: Zika virus infects pericytes in the choroid plexus and enters the central nervous system through the blood-cerebrospinal fluid barrier

doi: 10.1371/journal.ppat.1008204

Figure Lengend Snippet: a , CPs isolated from ZIKV-infected mice were subjected to immunostaining in a whole-mount choroid plexus assay with antibodies against ZO-1 and ZIKV-E. The top and stroma cell layer was imaged with a Zeiss LSM 710 Duo/LIVE 5 confocal microscope along the Z-axis, and the maximum Z-project image was created with Imaris software. b , HIBCPP Transwell model. HIBCPP cells were grown and differentiated into a monolayer in Transwell inserts. Fully developed HIBCPP Transwell inserts (determined by TEER) were placed in wells with various testing conditions (e.g., primary human brain pericytes or conditioned media). c . Fully developed HIBCPP barriers grown in Transwell inserts (n = 3–5 per group) were basolaterally exposed to ZIKV-infected HBVP cells (starting at 1 DPI). As a leakage control, a scratch was made on the HIBCPP cells fully grown in Transwell inserts. Samples from the apical and basolateral chambers were harvested every 24 hours, and the virus titers were determined. d , Fully developed HIBCPP barriers grown in Transwell inserts (n = 4–5 per group) were basolaterally exposed to ZIKV-infected HBVP cells (3 d.p.i) or fresh media spiked with ZIKV (1.0 x 10 5 pfu/well/mL). Twenty-four hours later, the cell supernatant in the apical camber was harvested and the virus amount was enumerated. e , HBVP (blue circles) and HIBCPP (red squares) cells grown on 12-well plates were infected with ZIKV (m.o.i. = 0.1) and then incubated for two days. The virus titers in the supernatants were enumerated with a virus titration assay. f , Expression of Axl and Tyro3 in HBVP and HIBCPP was determined by using realtime q-PCR and normalized to endogenous GAPDH expression.

Article Snippet: Primary human brain vascular pericytes (HBVPs) were obtained from ScienCell (Cat No. 1200) and maintained using the manufacturer’s protocol and their pericyte culture media with 2% FBS, 1 x pericyte growth supplement, and 1 x penicillin and streptomycin.

Techniques: Isolation, Infection, Immunostaining, Microscopy, Software, Control, Virus, Incubation, Titration, Expressing

Journal: Arthritis Research & Therapy

Article Title: Angiotensin II induces skin fibrosis: a novel mouse model of dermal fibrosis

doi: 10.1186/ar4028

Figure Lengend Snippet: Primary and secondary antibodies for immunofluorescence staining

Article Snippet: Next, cells were incubated at 4°C overnight with primary antibodies: goat anti-mouse VE-cadherin (Santa Cruz Biotechnology, Santa Cruz, CA, USA), and rabbit anti-mouse FSP1 (Abcam, Cambridge, MA, USA).

Techniques: Immunofluorescence