Journal: medRxiv

Article Title: Polymorphism in IFNAR contributes to glucocorticoid response and outcome in ARDS and COVID-19

doi: 10.1101/2022.03.10.22272123

Figure Lengend Snippet: (A ) STAT1 expression in the lung after 4-day culture in the presence of IFN beta with or without hydrocortisone (HC). ( B) pSTAT1 expression in the same specimens as in A. ( C) Example photomicrographs showing higher STAT2 expression in a TT patient than in a CT patient and the effect of HC on its nuclear translocation. Most STAT2 remains in the cytoplasm of the CT patients, whereas nuclear expression is prominent in the TT patient. Indicated insets are shown in the bottom row. Arrows. ( D ) Combined results of all patients noting that two CT samples are excluded in the data as the patients were already under glucocorticoid treatment at the time of sample acquisition. Ns, not significant; *P<0.05; **P<0.01; and ***P<0.001

Article Snippet: The first stage antibodies were anti-alpha chain of the IFN alpha/beta receptor (St John’s Laboratory STJ112765) that was used 1:2000 and 1:5000 and anti-Stat1 (1:400, 9175S), anti-pStat1(1:100, 9167S) and anti-Stat2 (1:200, 72604S) all from Cell Signalling.

Techniques: Expressing, Translocation Assay

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: Translational Oncology

Article Title: Anticancer Activity of a Broccoli Derivative, Sulforaphane, in Barrett Adenocarcinoma: Potential Use in Chemoprevention and as Adjuvant in Chemotherapy 1

doi:

Figure Lengend Snippet: Effect of SFN on BEAC cell survival. BEAC cells were cultured in the medium containing no SFN or various concentrations of SFN. Cells were harvested at different time points as indicated and proliferative potential was assessed by trypan blue exclusion and/or proliferation assay, based on the production of a yellow product (formazan) after reduction of a highly water-soluble tetrazolium salt by dehydrogenases in viable cells. The growth curves show the mean of three independent experiments, with SEM. (A) Barrett adenocarcinoma (FLO-1) cells treated with various concentrations of SFN. (B) BEAC (OE33) cells treated with various concentrations of SFN. (C) Photomicrograph of BEAC (FLO-1 and OE33) cells treated with 3 µM SFN for 72 hours. (D) Photomicrograph of normal diploid fibroblasts and primary normal esophageal epithelial cells (ScienCell Research Laboratories) treated with 3 µM SFN for 72 hours. (E) FLO-1 cells were treated with SFN for 48 hours, detached floating cells from the medium and the attached cells (by trypsinization) were collected separately and evaluated for number and viability using trypan blue exclusion. The number of cells detached after treatment with various concentrations of SFN is expressed as percent of untreated FLO-1 cells. “Total” represents the total number of detached cells whereas “Dead” reflects the fraction of dead cells in detached cell population. (F) Panel (I): FLO-1 cells were incubated with various concentrations of SFN for 48 hours, and the expression of caspase 8 was detected by Western blot analysis, using anti-caspase 8 mouse monoclonal antibody (Cell Signaling, Danvers, MA). Panel (II): Bar graph showing caspase 8 expression relative to β-actin.

Article Snippet: Normal primary human esophageal epithelial cells (HEEC) were purchased from ScienCell Research Laboratories (Carlsbad, CA) and have been described previously [ 30 ].

Techniques: Cell Culture, Proliferation Assay, Incubation, Expressing, Western Blot

Journal: PLoS ONE

Article Title: CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma

doi: 10.1371/journal.pone.0033005

Figure Lengend Snippet: ( A ) Human glioblastoma specimen immunostained for CXCL12 (brown) demonstrates expression in vascular endothelial cells. t = tumor cells, e = cross-section through tumor-associated capillary, and Scale bar = 25 µm. ( B ) HBMECs, in co-culture on Matrigel, express CXCL12 (red). Nuclei are counterstained blue with DAPI. Scale bar equals 25 µm. ( C ) Human brain micro-vascular endothelial cells cultured on Matrigel (Mat HBMEC) secrete CXCL12 into the media as determined by ELISA. Mat alone indicates results from Matrigel alone-conditioned media. N = 3. ** = p<0.005 as determined by two-tailed t -test. ( D ) Single cell suspensions from two different adult GBM patients (GBM1, GBM2) and cultured U87 cells express CXCR4 by western blotting. CXCR4 appears red and the actin loading control appears green.

Article Snippet: Primary human brain microvascular endothelial cells (HBMEC) were obtained from ScienCell, Carlsbad, CA).

Techniques: Expressing, Co-Culture Assay, Cell Culture, Enzyme-linked Immunosorbent Assay, Two Tailed Test, Western Blot, Control

Journal: PLoS ONE

Article Title: CXCL12 Mediates Trophic Interactions between Endothelial and Tumor Cells in Glioblastoma

doi: 10.1371/journal.pone.0033005

Figure Lengend Snippet: ( A ) Twenty-four hours after establishing a capillary-like network of mcherry-expressing HBMECs in Matrigel, eGFP-expressing U87 cells were added to the culture. Within 24 hours U87 cells were seen in physical contact with HBMECs. Scale bar = 50 microns. ( B ) The mean distances between U87 cells (500 to 800 cells) and HBMECs were calculated at different time points after the addition of the tumor cells to the HBMEC networks. There was a significant increase in co-localization (reduction in mean distance) within 24 hrs, which was maintained over a 72 hr period. * = p<0.05 as determined by one way ANOVA for the means of three separate experiments involving 500–800 measurements per experiment. ( C ) The distance between approximately 1000 eGFP-expressing U87 cells and mCherry fluorescent protein-expressing HBMECs in co-culture (24 hrs) was measured and the distribution was plotted as the percentage of total cells in 20 micron increments (black triangles). More than 50% of the total U87 cells in the culture were within 40 microns of an endothelial cell. A theoretical plot of a random distribution of cells is shown (open circles). ( D ) The distance between GFAP positive GBM cells and mCherry fluorescent protein-expressing HBMECs in co-culture were measured and the distribution was plotted as the percentage of total cells in 20 micron increments (black triangles). Error bars represent SEM from three independent experiments involving three different GBM isolates. Approximately 500 GBM (GFAP positive) cells were counted. Nearly 80% of the GFAP positive GBM cells in the culture were within 20 microns of an endothelial cell. A theoretical plot of a random distribution of cells is shown (open circles).

Article Snippet: Primary human brain microvascular endothelial cells (HBMEC) were obtained from ScienCell, Carlsbad, CA).

Techniques: Expressing, Co-Culture Assay