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

Journal: International Journal of Molecular Sciences

Article Title: Role of 14-3-3ζ in Platelet Glycoprotein Ibα-von Willebrand Factor Interaction-Induced Signaling

doi: 10.3390/ijms13055364

Figure Lengend Snippet: The S609A mutation inhibits activation of Src family kinases. ( a ) 1b9 or S609A cells were stimulated by ristocetin in the presence or absence of VWF, then solubilized and subjected to Western blot analysis with anti-Src and anti-phospho-Src family (pTyr416) antibody, respectively. The immunoblot is representative of 3 different experiments; ( b ) Quantitative data from 3 different experiments (mean ± SD) are demonstrated. The relative activation index of Src equals arbitrary quantitation of phospho-Src (pTyr416)/arbitrary quantitation of total Src.

Article Snippet: Anti-phospho-Src family (pTyr416) rabbit polyclonal antibody was from Cell Signaling Technology (Beverly, MA, USA).

Techniques: Mutagenesis, Activation Assay, Western Blot, Quantitation Assay

Journal: PLoS ONE

Article Title: MAPK Signaling Pathway Regulates p27 Phosphorylation at Threonin 187 as Part of the Mechanism Triggered by Early-Weaning to Induce Cell Proliferation in Rat Gastric Mucosa

doi: 10.1371/journal.pone.0066651

Figure Lengend Snippet: Animals were treated with 0.5% DMSO (control) or PD98059 at 300 µg/kg. Immunoblots and the respective densitometries are shown for p-ERK1/2, ERK1, p-Src and Src. Thirty µg of total protein were applied to each lane and each band is representative of one animal. β-actin was used as loading control. Relative densitometries as ratio of p-ERK/ERK1 and p-Src/Src are shown as fold change of control group and are presented by bars as means±SD (n = 3/group). *p<0.05 when comparing PD98059-treated group to control. Assays were conducted in duplicates.

Article Snippet: Samples were incubated overnight at 4°C with rabbit polyclonal antibodies to the signaling proteins ERK1 (1∶5000, sc-93, Santa Cruz Biotechnology) and phospho-Src (1∶300, #2101, Cell Signaling Technology), and to the cell cycle proteins cyclin E (1∶200, sc-481), CDK2 (1∶200, sc-163), cyclin D1 (1∶200, sc-718), CDK4 (1∶300, sc-601), p27 (1∶100, sc-528) (Santa Cruz Biotechnology), and phospho-p27 (T187, 1∶750, ab75908, ABCAM).

Techniques: Western Blot