Journal: Neuropharmacology

Article Title: Allosteric modulation of metabotropic glutamate receptor 4 activates IDO1-dependent, immunoregulatory signaling in dendritic cells

doi: 10.1016/j.neuropharm.2015.10.036

Figure Lengend Snippet: PI3K/Akt- and Src- but not G i -dependent signaling pathways are required for the production of IL-10 and TGF-β by DCs treated with ADX88178 and ADX104608. ( A ) Intracellular cAMP formation in DCs treated with mGluR4 PAMs. Cells were stimulated with forskolin (or left unstimulated as control) at 10 μM for 10 min in the presence or absence of ADX88178, ADX104608, and PHCCC at different concentrations. Ctrl, control. ( B ) Effect of PTX on the inhibition of IL-10 and induction of TGF-β by PHCCC in LPS-stimulated DCs. DCs treated with PHCCC at 30 μM were stimulated with LPS in the presence or absence of PTX at different concentrations and cytokine contents were measured in 24-h culture supernatants. ( C ) Effect of PTX on the induction of IL-10 and TGF-β by ADX88178 and ADX104608. DCs treated with ADX88178 or ADX104608 at 3 μM were left unstimulated or stimulated with LPS in the presence or absence of PTX at 0.2 μM and cytokine contents were measured in 24-h culture supernatants. ( D ) Kinetics of Akt and Src phosphorylation in WT DCs treated with ADX88178 at 3 μM. Lysates from drug-treated DCs (not subjected to LPS stimulation) were analyzed by sequential immunoblotting with antibody to phosphorylated Akt (pAkt) and anti-Akt or to phosphorylated Src (pSrc) and anti-Src. ( E ) Kinetics of Src phosphorylation in Grm4 −/− DCs treated as in D . ( F ) Effect of PI3K (LY294002; 25 μM) and Src (PP2; 5 μM) inhibitors on the induction of IL-10 and TGF-β by ADX88178 and ADX104608 in WT DCs. PP3 (5 μM) was used as PP2 negative control. DCs (not subjected to LPS stimulation) were treated with selective mGluR4 PAMs at 3 μM in the presence or absence of enzyme inhibitors and cytokine contents were measured in 24-h culture supernatants. Data ( A - C and F ), presented as means ± SD from triplicate samples, are shown for one representative experiment of three. In D and E , one experiment of two. * P < 0.05, ** P < 0.01, and *** P < 0.001.

Article Snippet: Drugs were added to the cultures 30 min before LPS, until recovering supernatants at 24 h. In selected experiments, LY294002 (inhibitor of PI3K; Cell Signaling Technology), PP2 (an Src inhibitor), and PP3 (negative control for the Src inhibitor; both from Tocris Bioscience) were also used at the final concentration of 25 (LY294002) or 5 (PP2 and PP3) μM with the same timing of PAMs.

Techniques: Protein-Protein interactions, Control, Inhibition, Phospho-proteomics, Western Blot, Negative Control

Journal: PLoS ONE

Article Title: Fibroblast Growth Factor 2 Causes G2/M Cell Cycle Arrest in Ras-Driven Tumor Cells through a Src-Dependent Pathway

doi: 10.1371/journal.pone.0072582

Figure Lengend Snippet: ( A ) Growth curves with FGF2 (10 ng/mL) and PP1 or PP2 (5 µM) added 1 hour before FGF2 (10 4 cells/cm 2 were plated). ( B ) Clonogenic assays of Y1 cells treated with FGF2 and/or PP1 (10 µM) or PP2 (5 µM) (added 30 minutes before FGF2) for 24 hours. Insets on the bottom show one representative clonogenic assay in the indicated conditions. Values are mean ± s.e.m. (n = 3). ( C ) Microphotographs for morphological analysis of Y1 cells under FCS or FGF2 stimulation for 48 hours, in the presence, where indicated, of PP2 (10 µM). ( D ) Y1 cells were synchronized in G0/G1 by serum starvation and stimulated with FCS (10%) and/or FGF2 (10 ng/mL) for 30 minutes in the presence of PP1 or PP2 (10 µM). Data are representative of four independent experiments. Phospho-Src (phosphorylated at Tyr416) and total Src were analyzed by immunoblot. Hprt was used as a loading control. ( E ) G0/G1-arrested Y1 cells were stimulated with FCS and/or FGF2 for 12 hours. Cells were pulse-labeled for 1 hour with [ 3 H]-thymidine before harvesting, and the amount of incorporated radioactivity was measured. FCS and FGF2 were added at time 0. PP1 or PP2 were added 1 hour before stimulation with FCS and/or FGF2. [ 3 H]-thymidine incorporation results are presented as the mean ± s.e.m. (n = 2). ( F ) Flow cytometry (FC) histograms show DNA content in Y1 cells after 24 and 48 hours of FCS and FGF2 treatment in G0/G1-starved cells. Cells were treated at time 0 and stained for DNA content and BrdU. FC histograms of DNA content (upper panels) and DNA/BrdU scatterplots (lower panels) of Y1 cells with or without PP2 (- PP2 and+PP2, respectively). Cells were stained with propidium iodide to assess DNA content. Biparametric flow cytometry analysis of DNA content and BrdU incorporation was performed on the same samples as described in the text. BrdU-positive cells were quantified by gates in the BrdU/DNA scatterplots. The upper panel shows the quantification of G0/G1, S and G2/M phases based on DNA content. The lower panel shows the quantification of G0/G1, S and G2/M phases based on DNA content versus BrdU labeling. Quantification of cell cycle phases, gated from the 2N to the 4N population only. Approximately 2×10 4 cells were analyzed. ( G ) Y1 cells were synchronized in G0/G1 by serum starvation and stimulated with FCS (10%) and/or FGF2 (10 ng/mL) for 30 minutes in the presence of PP1 or PP2 (10 µM). Data are representative of two independent experiments. phospho-ERK1/2 (phosphorylated at Thr202/Tyr204) and total ERK 1/2 were analyzed by immunoblot. Hprt was used as a loading control. Scale bar = 50 µm. SFM, serum-free media; FCS, fetal calf serum.

Article Snippet: Membranes were blocked for 1 hour in TBS-T buffer (150 mM sodium chloride, 50 mM Tris [pH 8], and 0.1% Tween 20) containing 5% nonfat milk and were then incubated with antibodies recognizing AKT (#9272), phospho-AKT Ser473 (#9272), ERK1/2 (#9102), phospho-ERK1/2 Thr202/Tyr204 (#9101L), phospho-Src Tyr416 (#2101), total Src (#2110) (all from Cell Signaling), and Hprt (sc-20975) (Santa Cruz).

Techniques: Clonogenic Assay, Western Blot, Labeling, Radioactivity, Flow Cytometry, Staining, BrdU Incorporation Assay