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: Molecular Biology of the Cell

Article Title: Receptor tyrosine kinase Met promotes cell survival via kinase-independent maintenance of integrin α3β1

doi: 10.1091/mbc.E15-09-0649

Figure Lengend Snippet: Loss of Met, but not its kinase activity, induces cell death. (A–D) Laminin-adherent PrECs treated with vehicle (DMSO), 0.2 μM PHA665752 (PHA), or 20 μM SU11274 (SU) for 72 h in starvation medium. (A) Met activity and total Met measured by immunoblotting of Met immunoprecipitates with anti-phosphotyrosine (P-Met) and Met antibodies. (B) Drug-treated cells imaged under phase-contrast light microscopy. (C) ATP levels and (D) caspase 3/7 activity measured in drug-treated cells compared with cells treated with 1 μM staurosporine (Str). Error bars are SD; n = 3. (E, F) Prostate epithelial cells isolated from Met fl/fl mice and Met loss induced by infection with virus expressing GFP (Ctl) or GFP-Cre (Cre). (E) Cells imaged under phase-contrast (left) or epifluorescence (right) microscopy 24 h after Cre infection. White dashed line marks the boundary between live and dead cells. (F) Met and full-length or cleaved caspase 3 measured by immunoblotting. (G, H) Prostate epithelial cells isolated from Met fl/fl mice crossed to Cre-ER TM mice and Met knockout induced by treatment with vehicle (EtOH) or 1.5 μM tamoxifen (Tmx). (G) Cells imaged under phase-contrast light microscopy before treatment (0 h) or 48 h later. (H) Met, full-length caspase 3, Bcl-xL, and GAPDH measured by immunoblotting.

Article Snippet: PrECs adherent to endogenous laminin matrix and starved for 24 h were treated with 1 μM staurosporine (Promega, Madison, WI), 10–20 μM SU11274 (Calbiochem, Ontario, Canada), or 0.1–1 μM PHA665752 (provided by James Christensen, Pfizer, La Jolla, CA).

Techniques: Activity Assay, Western Blot, Light Microscopy, Isolation, Infection, Expressing, Microscopy, Knock-Out