Journal: Clinical Cancer Research

Article Title: Increased TGF-α as a Mechanism of Acquired Resistance to the Anti-EGFR Inhibitor Cetuximab through EGFR–MET Interaction and Activation of MET Signaling in Colon Cancer Cells

doi: 10.1158/1078-0432.ccr-13-0423

Figure Lengend Snippet: Figure 4. PHA655752 restores sensitivity of GEO-CR and SW48- CR cells to cetuximab. A and C, GEO, GEO-CR (A), SW48, and SW48-CR (C) cells were treated with increased concentrations of PHA665752 (0.01–10 nmol/L) for 96 hours and evaluated for cell proliferation by MTT staining, as described in Materials and Methods. The results are the average SD of three independent experiments, each done in quadruplicate. B and D, GEO-CR (B) and SW48-CR (D) cells were treated with 2 doses of PHA665752 (0.01 or 0.05 nmol/L for GEO-CR and 5 or 10 nmol/L for SW48-CR), with increasing concentrations of cetuximab (0.01–10 mg/mL) or with a combination of both drugs for 96 hours and evaluated for cell proliferation by MTT staining, as described in Materials and Methods. The results are the average SD of three independent experiments, each done in quadruplicate. E and F, GEO-CR (E) and SW48-CR (F) cells were treated with PHA665752, cetuximab, or their combination at the indicated concentrations for 24 hours. The cell lysates were assayed by Western blotting with the indicated antibodies, as described in Materials and Methods. G and H, GEO-CR (G) and SW48-CR (H) cells were treated with PHA665752, cetuximab, or their combination at the indicated concentrations and analysis of in vitro cell migration was performed as described in Materials and Methods.

Article Snippet: PHA665752, a selective MET tyrosine kinase inhibitor (TKI), was purchased from Santa Cruz Biotechnology.

Techniques: Staining, Western Blot, In Vitro, Migration

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