Journal: Slas Discovery

Article Title: Development of a 3D Tissue Culture–Based High-Content Screening Platform That Uses Phenotypic Profiling to Discriminate Selective Inhibitors of Receptor Tyrosine Kinases

doi: 10.1177/1087057116657269

Figure Lengend Snippet: c-Met–dependent phenotypic changes. ( A ) Inhibition of hepatocyte growth factor (HGF)–induced invasion in 3D-cultured PC-3 cells by function blocking c-Met Fab antibody 5D5 (Genentech, South San Francisco, CA) and stimulation of invasion by the crosslinking and activating bivalent IgG isoform. Principal components analysis (PCA) trained on unstimulated and stimulated control, PC0 shown and scaled as Z score to unstimulated control. Results are shown as means of quadruplicate wells and standard deviations. ( B ) 5D5 Fab (triangles) causes a shift in phenotype from stimulated (empty circles) back to unstimulated control (filled circles). Point size of markers for Fab treatment increases with concentration (seven concentrations; range, 0.316–316 nM). Individual data points are shown as a 3D scatterplot with PC0, PC1, and PC2.

Article Snippet: Results showed that PHA-665752 and the Genentech 5D5 Fab potently inhibited phosphorylation of c-Met but not EGFR phosphorylation.

Techniques: Inhibition, Cell Culture, Blocking Assay, Concentration Assay

Journal: Frontiers in Molecular Neuroscience

Article Title: Autism Spectrum Disorder Risk Factor Met Regulates the Organization of Inhibitory Synapses

doi: 10.3389/fnmol.2021.659856

Figure Lengend Snippet: Hepatocyte growth factor (HGF) increases gephyrin clustering in vitro . Hippocampal neurons were left untreated or were treated with DMSO, HGF, or PHA-665752. Representative images of gephyrin- or MAP2-stained hippocampal neurons are shown in the absence (A–A”) or presence (B–B”) of HGF. Overview images are shown in ( A,B ; scale bar 20 μm) while insets are enlarged in ( A’,A”,B’,B” ; scale bar 10 μm). Panels (A’,B’) depict confocal images, (A”,B”) processed images for the quantification of gephyrin cluster (red) densities within masks of MAP2-positive dendritic segments (25 μm). ( C) Quantification of gephyrin cluster densities. One-way ANOVA and Tukey’s multiple comparison test F (4,364) = 0.7245. CTR: n = 75, HGF: n = 71, DMSO n = 78, PHA n = 73, PHA/HGF n = 72. *** p < 0.0001, error bars SEM. (D,E) Untreated hippocampal neurons (D) or treated with HGF (E) were stained for phosphorylated p70S6K (pS6K, scale bar 20 μm). Insets are enlarged in ( D’,E’ ; scale bar 10 μm) for determination of mean fluorescence intensities of phosphorylated p70S6K signals within a mask of MAP2-positive cell bodies. Dashed outlines mark the area of the cell bodies. (F) Quantification of phosphorylated p70S6K intensities. CTR n = 57, DMSO n = 83, HGF n = 83, PHA n = 68, PHA/HGF n = 77. One-way ANOVA and Tukey’s multiple comparison test F (4,363) = 3.369. *** p < 0.0001, error bars: SEM.

Article Snippet: Inhibitors were dissolved in dimethylsulfoxide (DMSO), HGF (Merck KGaA, Darmstadt, Germany) in PBS and added to hippocampal neurons (DIV 9) to final concentrations of 50 ng/ml for HGF, 200 nm for mTOR inhibitor rapamycin (Merck), and 1 μM PHA-665752 (Merck KGaA, Darmstadt, Germany).

Techniques: In Vitro, Staining, Fluorescence

Journal: Stem Cell Research & Therapy

Article Title: Paracrine stimulation of perinatal lung functional and structural maturation by mesenchymal stem cells

doi: 10.1186/s13287-020-02028-4

Figure Lengend Snippet: Growth factors were not responsible for the stimulating effect of MSC-CM on Na + transport. FDLE cells were subjected to MSC-CM or control medium for 24 h with or without the respective growth receptor inhibitor. Data are displayed as boxes and whiskers with the 10–90 percentiles, mean (+), and median (horizontal line). Statistical differences among groups were analyzed with an ANOVA and Tukey’s post hoc test. a Inhibition of VEGF-R with Axitinib ( n = 19–23, 2 IE; *** p < 0.001), b BMP-R with K02288 ( n = 22–24, 2 IE; *** p < 0.001), and c PDGF-R with AG-1296 ( n = 18–22, 2 IE; *** p < 0.001) did not affect Na + transport in MSC-CM-treated and control cells. d The EGF-R inhibitor AG-1478 ( n = 19–24, 2 IE; *** p < 0.001), e the TGF-β-R inhibitor SB431542 ( n = 21–24, 2 IE; * p < 0.05; *** p < 0.001), f the FGF-R inhibitor FIIN-2 ( n = 16–24, 2 IE; * p < 0.05; ** p < 0.01; *** p < 0.001), and g the HGF-R (c-met) inhibitor PHA665752 ( n = 64–67, 6 IE; ** p < 0.01; *** p < 0.001) reduced ∆ I amil in control and MSC-CM-treated cells. Inhibition did not prevent the stimulating effect of MSC-CM. □ control, ■ MSC-CM

Article Snippet: The inhibitors of the VEGF receptor (Axitinib, 10 μM), the bone morphogenetic protein receptor (BMP-R; K02288, 1 μM), the platelet-derived growth factor receptor (PDGF-R; AG-1296, 10 μM), the EGF receptor (EGF-R; AG-1478, 1 μM), the transforming growth factor β receptor (TGF-β-R; SB431542, 1 μM), the FGF receptor (FGF-R; FIIN-2, 1 μM), and the HGF receptor c-met (PHA665752, 1 μM) were purchased from Biomol (Hamburg, Germany).

Techniques: Control, Inhibition