Journal: PloS one

Article Title: Differential roles of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in the regulation of S115 breast cancer cell growth.

doi: 10.1371/journal.pone.0049970

Figure Lengend Snippet: Figure 1. FGFR1, 2 and 3 expression in FGFR-silenced S115 cells (sh cells). Cells were grown in DMEM containing 4% iFBS and 10 nM testosterone. RNA and protein were extracted from three independently cultured sub-confluent cell plates and analyzed using qRT-PCR and immunoblotting, respectively. A) FGFR1-3 mRNA expression in the five original FGFR-silenced cell pools (shR1B, shR1D, shR2IA, shR2ADG and shR3B) compared to the expression level in control cells (shLacZ). FGFR mRNA expression is normalized to cyclophilin B expression. The statistical differences between mRNA levels were tested by non-parametric Mann-Whitney U-test, * P,0.05, ** P,0.01. B) Overall relative FGFR mRNA expression level in sh cells. C) Relative FGFR1IgIIIb/c and FGFR2IgIIIb/c mRNA expression in cells that were chosen for further studies (shR1B, shR2IA and shR3B). D) FGFR protein levels in shR1B, shR2IA and shR3B cells. Immunoprecipitation of FGFR1 was performed using 150 mg of whole cell lysates. Detection of FGFR2 and FGFR3 by western blotting was performed using 20 mg of whole cell lysates. FGFR1 expression in shR1, shR2 and shR3 cells is normalized to the protein expression in shLacZ cells. FGFR2 and FGFR3 expression in shR1, shR2 and shR3 cells are normalized to b-actin expression and thereafter to expression in shLacZ. A representative immunoblot of three independent experiments is shown. doi:10.1371/journal.pone.0049970.g001

Article Snippet: FGFR2 Transfections The FGFR2 expression constructs SC112364 and SC111932 (here named pFGFR2IgIIIb and pFGFR2IgIIIc, respectively) were purchased from Origene (OriGene Technologies, Inc, Rockville, MD). shLacZ cells were transfected with FGFR2IgIIIb or FGFR2IgIIIc plasmids or with the vector control pCMV6-Neo using Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions.

Techniques: Expressing, Cell Culture, Quantitative RT-PCR, Western Blot, Control, MANN-WHITNEY, Immunoprecipitation

Journal: PloS one

Article Title: Differential roles of fibroblast growth factor receptors (FGFR) 1, 2 and 3 in the regulation of S115 breast cancer cell growth.

doi: 10.1371/journal.pone.0049970

Figure Lengend Snippet: Figure 7. Overexpression of FGFR2IgIIIb and FGFR2IgIIIc in shLacZ cells. A) FGFR2 mRNA levels (relative to control-transfected cells (pCMV6-neo)) in FGFR2IgIIIb and FGFR2IgIIIc-transfected cells were analyzed by qRT-PCR. mRNA levels measured 24 h post-transfection are presented in a logarithmic scale. B) FGFR2 protein levels 24 h post-transfection shown by western blotting. C) Relative FGFR1 mRNA expression in FGFR2IgIIIb and FGFRIgIIIc-overexpressing cells 24 h post-transfection. The experiment was repeated twice with similar results. doi:10.1371/journal.pone.0049970.g007

Article Snippet: FGFR2 Transfections The FGFR2 expression constructs SC112364 and SC111932 (here named pFGFR2IgIIIb and pFGFR2IgIIIc, respectively) were purchased from Origene (OriGene Technologies, Inc, Rockville, MD). shLacZ cells were transfected with FGFR2IgIIIb or FGFR2IgIIIc plasmids or with the vector control pCMV6-Neo using Lipofectamine 2000 (Invitrogen) according to the manufacturers’ instructions.

Techniques: Over Expression, Control, Transfection, Quantitative RT-PCR, Western Blot, Expressing

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were transfected with wt FGFR3 or activating FGFR3 mutants (N540K, G380R, R248C, Y373C, K650M, K650E), and analyzed for the indicated molecules by WB 48 hours later. The levels of ERK phosphorylation vary among the tested mutants, reflecting the different strength of FGFR3 activation by each particular mutation . K508M - kinase inactive FGFR3 mutant. GFP and empty vectors serve as transfection controls. (B) LRP6 phosphorylation at Thr1572 caused by highly activating FGFR3 mutants R248C and K650E. (C) Cells were transfected with the indicated FGFR3 vectors together with Topflash reporter vectors, treated with WNT3a and analyzed for luciferase activity. Data represent an average from three transfections (each measured twice), with the indicated standard deviations. A logarithmic scale of the y -axis is necessary to express the massive Topflash activation in WNT3a-treated cells expressing activating FGFR3 mutants (* p <0.001; Student’s t -test; compared to wt FGFR3). Results are representative of four experiments. (D) Cells were transfected with wt FGFR2 or activating FGFR2 mutants (S252W, P253R, C342R, C342Y, Y375C), and analyzed for the indicated molecules by WB. Note the significant ERK and LRP6 phosphorylation caused by C342R, C342Y and Y375C mutants, which correlates with increased basal (E; upper graph) and WNT3a-induced (E; lower graph) β-catenin activity, evidenced by Topflash experiment. Results are representative for three experiments (* p <0.001; Student’s t -test; compared to wt FGFR2).

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Transfection, Phospho-proteomics, Activation Assay, Mutagenesis, Luciferase, Activity Assay, Expressing

Journal: PLoS ONE

Article Title: Receptor Tyrosine Kinases Activate Canonical WNT/β-Catenin Signaling via MAP Kinase/LRP6 Pathway and Direct β-Catenin Phosphorylation

doi: 10.1371/journal.pone.0035826

Figure Lengend Snippet: (A) RCS cells were treated for indicated times with FGF2 (10 ng/ml) in the presence of heparin (1 µg/ml), and analyzed for β-catenin phosphorylation at Tyr142 by WB (arrow). (B) HEK293 cells were transfected with wt FGFR2 or its activating mutant Y375C, and analyzed for indicated molecules 48 hours later. Note the increased β-catenin phosphorylation at Tyr142 (arrow). (C) Active recombinant FGFR3, FGFR2, TRKA and EGFR were subjected to a cell-free kinase assay with recombinant β-catenin as a substrate. Samples with ATP or kinase omitted serve as controls for kinase reaction.

Article Snippet: For the recombinant RTK kinase assays, the reactions were carried-out with 400 ng of recombinant FGFR2, FGFR3, TRKA or EGFR (SignalChem, Richmond, Canada) and 400 ng of recombinant β-catenin (Abcam, Cambridge, MA, USA) in 50 μl of kinase buffer in the presence of 50 μM ATP for 60 minutes at 30°C.

Techniques: Phospho-proteomics, Transfection, Mutagenesis, Recombinant, Kinase Assay

Journal: International Journal of Clinical and Experimental Pathology

Article Title: CXCL5 contributes to the tumorigenicity of cervical cancer and is post-transcriptionally regulated by miR-577

doi:

Figure Lengend Snippet: CXCL5 is up-regulated in cervical cancer tissues and cell lines. IHC staining was performed to analyze CXCL5 positive staining in cervical cancer tissues and para-carcinoma tissues (A: left: magnification 40×; right: magnification 200×) and quantitative analysis (B). Western blot was used to measure CXCL5 protein expression in human normal cervical squamous cell line Ect1/E6E7 and cervical cancer cell lines Hela and SiHa (C and D). *P < 0.05. CC, cervical cancer.

Article Snippet: Human normal cervical squamous cell line Ect1/E6E7 (CHI Scientific, Inc., Maynard, MA, USA) and cervical cancer cell lines Hela and SiHa (ATCC, USA) were maintained in DMEM; (Gibco, USA) containing 10% FBS (Gibco, USA) with 5% CO 2 at 37°C. miR-Con, miR-577 mimics, miR-577-Mut, and overexpressed CXCL5 plasmids were purchased from GenePharm (Shanghai, China) and transfected into Hela and SiHa cells using lipofectamine 2000 (Invitrogen).

Techniques: Immunohistochemistry, Staining, Western Blot, Expressing