Immunostaining of NR4A2 in gastric adenocarcinoma.

<p><b>A-B</b>: NR4A2 immunoreactivity in normal oxyntic mucosa showing strong intensity in scattered single cells (neuroendocrine cells) and weaker staining intensity in the other epithelial cells. <b>C-F</b>: NR4A2 immunoreactivity in gastric adenocarcinomas of intestinal (C-D) and diffuse (E-F) type, showing a general staining in tumor cells with mixed nuclear or cytoplasmic localization and variable intensities. (A, C, E at x200 magnification, with boxes representing B, D and F at x400 magnification).</p

NR4A2 activates NBRE promoter elements.

<p><b>A</b>: Gastrin-induced NBRE-luc activation. Data represent one of two biological replicas. <b>B</b>: The effect of NR4A2 siRNA on gastrin-induced NBRE activation. Data represent mean ± SEM of four biological replicas (** p<0.01, * p=0.1). <b>C-D</b>: Effect of specific inhibitors of PKA (H-89, 10µM), PI3K (LY 294002, 10µM) or PKC (GF 109203x, 3.5µM) on (<b>C</b>) gastrin-induced NR4A2 gene expression and (<b>D</b>) gastrin-induced NBRE activation. Data represent one of three biological replicas; mean ± SD of six technical replicas.</p

Negative regulation of gastrin-induced NR4A2 expression.

<p><b>A</b>: AGS-G_R cells transfected with NR4A2-luc and ICER expression plasmids or empty vector. Cells were treated with gastrin for 6 h prior to measurement of NR4A2 activity. Data shown represent mean ± SEM of five biological replicas (** p<0.03, * p = 0.06). <b>B</b>: AGS-G_R cells transfected with NBRE-luc and ICER expression plasmid or empty vector and treated with gastrin for 4 h prior to measurement of NBRE activity. Data shown represent mean ± SEM of four biological replicas (** p<0.03). <b>C</b>: AGS-G_R cells were transfected with pZfp36l1 expression plasmid or empty vector and treated with gastrin (5 nM) NR4A2 mRNA expression was measured by qRT-PCR. Data shown represent one of three biological replicas; mean ± SD of three technical replicas is shown. <b>D</b>: Cyclin L1 represents one of three control genes examined.</p

NR4A2 suppresses gastrin-induced migration and invasion.

<p><b>A</b>: Real-time cell migration monitored (0-24 h) in AGS-G_R cells transfected with siNR4A2 or siCtr, with or without gastrin treatment (10 nM). Results show one representative of three biological replicas; mean ±SD of three technical replicas. <b>B</b>: Invasion assay with AGS-G_R cells transfected with pCMX-NR4A2 or pCMX (control) was performed in 24-well plates containing 8-µm pore Matrigel-coated inserts (with or without 0.3 nM gastrin). Cells invading the lower surface of the membrane were stained with Reastain Quick-Diff reagents and total numbers of cells in 5 fields per membrane were counted. The mean of three independent experiments is shown.</p