Journal: The FASEB Journal

Article Title: Resident mesenchymal vascular progenitors modulate adaptive angiogenesis and pulmonary remodeling via regulation of canonical Wnt signaling

doi: 10.1096/fj.202000629r

Figure Lengend Snippet: FIGURE 5 Disrupted Wnt signaling in ABCG2pos COPD MVPC disrupts MVEC function. A, Heatmap for differentially expressed genes in MVPC from COPD vs non-diseased lungs (color scale shown at the top right) B and C, Validation of representative genes expressed in COPD ABCG2pos lung MVPC, compared to control, enriched in GO categories of B, Wnt signaling and C, actin binding, contractility, and migration. The mean of combined patient samples per group as well as results for individual samples are presented. n = 3-6 patient samples per group. D, Western blot analysis of Dkk1, Wnt5a and Slit2 protein expression. n = 3, 3. Data presented as the mean ± standard error of mean (SEM). E, Cocultures of pulmonary MVEC and MVPC were analyzed for the effect of control or COPD MVPC on barrier function following injury. MVPC were plated on a monolayer of MVEC at a ratio of 3:1. The groups underwent no injury or an electrical wounding injury using the ECIS system. The presence of COPD MVPC following injury decreased the rate at which barrier formation is recovered. Quantitation of normalized resistance at indicated time points (Δ) was presented in bar graph format. Data presented as mean (±SEM). Controls included MVEC alone and uninjured MVEC. n = 4. F, Murine WT and Wnt activated βOE MVPC were analyzed by PCR to examine the expression of angiogenic transcripts identified as different between control and COPD samples. n = 3, 3. All amplification was normalized to a housekeeping gene and the results presented as mean fold change over control. Data presented as mean ± SEM. G. Representative summary of murine and human PCR data

Article Snippet: Human lung MVECs (Lonza, Walkersville) were plated at a concentration of 112 500 cells per well on gelatin coated 8W1E PET ECIS culture ware arrays (Applied Biophysics, Troy) overnight to achieve confluence.

Techniques: Biomarker Discovery, Control, Binding Assay, Migration, Western Blot, Expressing, Quantitation Assay, Amplification

Journal: bioRxiv

Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

doi: 10.1101/2020.08.02.233064

Figure Lengend Snippet: A . ECIS trace for untreated and severe cocktail-treated wells. Blue lines represent concentrations of ruxolitinib. B, C . Protection of endothelial barrier integrity with active compounds. Data were averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds (n=5).

Article Snippet: Prior to use, 96-well ECIS plates (Applied Biophysics, 96W20idf PET) were pre-treated with 10mM L-cysteine (Sigma-Aldrich, C7352-25G) and then coated with fibronectin (gibco, PHE0023).

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Functional immune mapping with deep-learning enabled phenomics applied to immunomodulatory and COVID-19 drug discovery

doi: 10.1101/2020.08.02.233064

Figure Lengend Snippet: A . Syk, c-Met and PI3K inhibitors rescue the severe COVID-19 specific cytokine storm high-dimensional phenoprint (perturbed state) to the healthy phenoprint (target state). B . Example images of target and perturbed cell populations for the cytokine storm and SARS-CoV2 viral models. C . Infection of HRCE yielded a phenoprint against the mock-infected target population with an assay z-factor of 0.43 for the separation in on-perturbation score for the mock and infected populations. D-F . Projections of compound response in the context of the perturbation vector generated in SARS-CoV-2-infected HRCE, Vero, and Calu3 cells. Off-perturbation values clipped at 50 for visualization. G . Compound impact on endothelial barrier function as quantified by ECIS assay. Values are normalized from 0 (cytokine storm cocktail-treated wells) to 100 (mock-treated wells). Data was averaged over a 12-minute window at hour 12 of ECIS measurement to visualize concentration response curves for the indicated compounds. H . Infection rate as determined by SARS-CoV-2 nucleocapsid antibody staining of infected HRCEs treated with the denoted compounds. I . Plot of efficacious molecules by hit-scores in SARS-CoV-2 HRCE assay vs cytokine storm assay. Orange circles denote molecules registered in interventional COVID-19 clinical trials at the time of submission. Dotted lines presented as a visual guide depicting a hit score of 0.6.

Article Snippet: Prior to use, 96-well ECIS plates (Applied Biophysics, 96W20idf PET) were pre-treated with 10mM L-cysteine (Sigma-Aldrich, C7352-25G) and then coated with fibronectin (gibco, PHE0023).

Techniques: Infection, Plasmid Preparation, Generated, Concentration Assay, Staining, Clinical Proteomics

Journal: Cell Death and Differentiation

Article Title: Proteasome inhibition and oxidative reactions disrupt cellular homeostasis during heme stress

doi: 10.1038/cdd.2014.154

Figure Lengend Snippet: Proteasome inhibition sensitizes cells for heme toxicity. ( a ) Monolayer resistance of confluent Hmox1 (+/+) MEF cells was measured with an ECIS instrument during incubation of the cells with or without heme (10 μ M), bortezomib (100 pM), or the combination of heme+bortezomib. Data represent mean±S.D. of four biologic replicates. At the end of the experiment, cells were fixed and stained for fluorescence microscopy with DAPI (yellow: nuclei) and Alexa 488 phalloidin (cyan: actin cytoskeleton). ( b ) Hmox1 (+/+) MEF cells were treated with a range of heme concentrations in the presence or absence of bortezomib (30 pM and 100 pM). After 12 h, cellular ATP was measured with a luminescence assay. Data are normalized to the respective control and represent mean±S.D. of six biologic replicates

Article Snippet: To support this hypothesis, we studied whether proteasome inhibition by bortezomib could make the more heme-resistant Hmox1 (+/+) MEF cells more sensitive to the impact of heme toxicity. shows monolayer integrity measured with an Electric Cell-substrate Impedance Sensing (ECIS) system (ibidi GmbH, Planegg/Martinsried, Germany) instrument over time as well as end point fluorescence images of Hmox1 (+/+) MEF cells after stimulation with bortezomib, heme (10 μ M), or bortezomib+heme.

Techniques: Inhibition, Incubation, Staining, Fluorescence, Microscopy, Luminescence Assay, Control