Journal: Molecular and Cellular Biology

Article Title: Phosphorylation Regulates FOXC2-Mediated Transcription in Lymphatic Endothelial Cells

doi: 10.1128/MCB.01387-12

Figure Lengend Snippet: Phosphorylation regulates FOXC2-mediated transcription in primary LECs. (A) Immunofluorescent staining for Myc (green), lymphatic marker PROX1 (red), and DNA (blue) of LECs transduced with adenoviruses expressing wild-type Myc-FOXC2, phosphorylation-deficient mutant Myc-pmFOXC2, or control bacterial β-galactosidase (LacZ). Note that wild-type and mutant FOXC2 have similar expression levels and subcellular localization. Bars, 20 μm. (B) Phosphorylation regulates FOXC2 transcriptional activity. Gene expression profiling was performed on the adenovirus-transduced LECs shown in panel A. Genes whose expression changed >2-fold in response to the loss of FOXC2 phosphorylation (FDR < 0.01) are shown in orange (upregulated) and purple (downregulated) in the Volcano plot of significance against the fold change in gene expression. Vertical dotted lines mark the 2-fold change limits. (C) RT-PCR validation of the gene expression profiling results. Genes upregulated or downregulated >2-fold in response to the loss of FOXC2 phosphorylation are shown in orange and purple, respectively; genes affected <2-fold are shown in gray. No change in FOXC2 expression reflects equally efficient cell transduction with Ad-Myc-FOXC2 and Ad-Myc-pmFOXC2. The data are presented as log2-transformed fold change in gene expression normalized to a housekeeping gene (GAPDH). Horizontal dotted lines mark the 2-fold change limits. Shown are the means and standard deviations of triplicate determinations in a single experiment representative of two independent experiments. (D) Heat map representation of the differences in gene expression in response to the loss of FOXC2 phosphorylation. The left heat map shows expression levels of 57 of 59 genes downregulated >2-fold (FDR < 0.01) in Ad-Myc-pmFOXC2-transduced LECs compared to Ad-Myc-FOXC2-transduced LECs. The right heat map shows expression levels of 57 out of 88 genes upregulated >2-fold (FDR < 0.01) in the same cells. Three biological replicates are shown for each condition. The color key at the lower left corresponds to the mean-centered, arctan-transformed log2-fold change in gene expression falling within the range from −π/2 to π/2. Blue denotes genes with relative decreased expression; red denotes genes with relative increased expression.

Article Snippet: For immunofluorescence staining, we used mouse anti-Myc (clone 9E10; Santa Cruz Biotechnology), rabbit anti-Myc-Tag (clone 71D10; Cell Signaling Technology, used for transgene detection in vivo ), rat anti-mouse PECAM-1 (BD Pharmingen), rat anti-mouse FOXC2 ( 19 ), sheep anti-human FOXC2 (R&D Systems), and goat anti-human PROX1 (R&D Systems).

Techniques: Staining, Marker, Transduction, Expressing, Mutagenesis, Activity Assay, Reverse Transcription Polymerase Chain Reaction, Transformation Assay

Journal: Cancer Science

Article Title: Role of tumor‐associated lymphatic endothelial cells in metastasis: A study of epithelial ovarian tumor in vitro

doi: 10.1111/j.1349-7006.2009.01436.x

Figure Lengend Snippet: The culture and identification of LYVE‐1+ cells isolated from epithelial ovarian tumor. LYVE‐1+ cell growth was recorded under phase‐contrast microscope. Identification of second‐generation LYVE‐1+ cells by lymphatic endothelial cell marker Prox‐1 (nuclear) and LYVE‐1 (membrane). Immunofluorescence shows LEC markers Podoplanin and VEGFR‐3, and pan‐endothelial cell marker CD31 (all located on membrane) were all expressed by these cells.

Article Snippet: Rabbit antihuman LYVE‐1 Ab was from Upstate Biotechnology (Charlottesville, VA, USA), goat antihuman Prox‐1 Ab and human recombination VEGF‐C were from R&D Systems (Minneapolis, MN, USA), and mouse antihuman VEGFR‐3 Ab was from Chemicon (Temecula, CA, USA).

Techniques: Isolation, Microscopy, Marker, Membrane, Immunofluorescence