Differential expression of FILIP1L protein in various human cancer cell lines.

<p>Immunoblot analysis for FILIP1L in the same cells utilized in Figure 1. GAPDH blot is shown as the loading control. The result is representative of three independent experiments. </p

Increase of cell invasion following FILIP1L knockdown in various FILIP1L-high-expressing, low-invasive cancer cell lines.

<p>MCF7 breast (<i>B</i>), NCM460 colon (<i>C</i>), H661 lung (<i>D</i>) and Capan-1 pancreatic (<i>E</i>) cancer cell lines were transfected with either non-targeting or <i>FILIP1L</i> siRNA. <i>A</i>, Immunoblot analysis for FILIP1L in the transfected cells. GAPDH blot is shown as the loading control. <i>B</i>-<i>E</i>, Transfected cells were subject to Matrigel cell invasion assay 48 h after transfection. The same experimental procedures were followed as described in Figure 6. The result is representative of two independent experiments. </p

Inverse correlation of <i>FILIP1L</i> expression with <i>FILIP1L</i> promoter methylation in various human cancer cell lines.

<p><i>A</i>-<i>D</i>, DNA methylation status of the CpG island in the <i>FILIP1L</i> promoter from the same cell lines utilized in Figure 1 was analyzed by Sequenom® EpiTYPER Mass Array. Mass Array results are shown as the average overall methylation for the analyzed 21 CG sites out of total 59 CG sites in the CpG island of the <i>FILIP1L</i> promoter. Error bars indicate SEM (<i>n</i> = 3). <i>E-H</i>, A significant inverse correlation of the DNA methylation status of the CpG island in the <i>FILIP1L</i> promoter with <i>FILIP1L</i> mRNA expression (each <i>P</i> value was calculated by Spearman’s rank correlation method). y axis: percent methylation of the average overall methylation for all 21 CG sites shown in sections <i>A-D</i> was used. x axis: standardized <i>FILIP1L</i> mRNA expression shown in Figure 1 was used.</p

Inhibition of cell invasion following FILIP1L expression in various FILIP1L-low-expressing, highly-invasive cancer cell lines.

<p>The same cell lines used in Figure 4 were transfected with control, wild-type <i>FILIP1L</i> or <i>FILIP1LΔC103</i> cDNA. <i>A</i>, Immunoblot analysis for FILIP1L in the transfected cells. GAPDH blot is shown as the loading control. <i>B</i>-<i>E</i>, Transfected cells were subject to Matrigel cell invasion assay 24 h after transfection. The same experimental procedures were followed as described in Figure 6. The result is representative of two independent experiments. </p

Association of reduced methylation in the <i>FILIP1L</i> promoter with restoration of FILIP1L expression in various cancer cells following treatment with a DNA demethylating agent.

<p>DNA methylation status of the CpG island in the <i>FILIP1L</i> promoter from the same cells used in Figure 4 was analyzed by Sequenom® EpiTYPER Mass Array. Mass Array results are shown as described in Figure 3A-D. The result is an average of two independent experiments. <i>P</i> values are derived from comparison between DMSO-treated control and each DAC-treated experiment.</p

Inverse correlation of FILIP1L expression with the invasive potential of various human cancer cell lines.

<p><i>A</i>-<i>D</i>, Matrigel cell invasion assay for the same cells utilized in Figure 1. Matrigel invasion was measured using the BD BioCoat Tumor Invasion System as described in <i>Materials and Methods</i>. The <i>y</i> axis represents a percent change over serum-free control. Error bars indicate SEM (<i>n</i> = 4). The result is representative of three independent experiments. <i>E-H</i>, A significant inverse correlation of the <i>FILIP1L</i> mRNA expression with invasiveness of the cells (each <i>P</i> value calculated by Spearman’s rank correlation method). y axis: invasiveness of the cells as a percent change over serum-free control shown in sections <i>A-D</i> was used. x axis: standardized <i>FILIP1L</i> mRNA expression shown in Figure 1 was used. </p

Restoration of <i>FILIP1L</i> expression in various cancer cells following treatment with a DNA demethylating agent or a histone deacetylase inhibitor.

<p>qRT-PCR analysis for <i>FILIP1L</i> conducted on cDNA from BT-549 breast (<i>A</i>), HT-29 colon (<i>B</i>), H1299 lung (<i>C</i>) and MIA PaCa-2 pancreatic (<i>D</i>) cancer cell lines treated with either 5-aza-2’-deoxycytidine (DAC) or Trichostatin A (TSA). Values 0.1, 1 or 10 in the <i>x</i> axis indicate the concentration of DAC and TSA in μM. The <i>y</i> axis represents fold change of each reagent-treated cell type over DMSO-treated control cells, where each value was standardized with the housekeeping gene <i>hRPL7</i>. Error bars indicate SEM (<i>n</i> = 3). The result is an average of two independent experiments. <i>P</i> values are derived from comparison between DMSO-treated control and either DAC- or TSA-treated experiments. NS indicates not significant. </p

Differential expression of <i>FILIP1L</i> mRNA in various human cancer cell lines.

<p>qRT-PCR analysis for <i>FILIP1L</i> conducted on cDNA from human breast (<i>A</i>), colon (<i>B</i>), lung (<i>C</i>) and pancreatic (<i>D</i>) cancer cell lines. The <i>y</i> axis represents <i>FILIP1L</i> mRNA expression which was standardized with the housekeeping gene <i>hRPL7</i>. Error bars indicate SEM (<i>n</i> = 3). The result is an average of three independent experiments. </p

Deletion of <i>Men1</i> in the parathyroid hormone-secreting cells increases ARC mRNA expression.

<p>A) Parathyroid tissue (inside blue circle) from 12 m old PTH-Cre; Men1 f/f mouse subsequently subjected to laser microdissection. B) qRT-PCR for ARC in parathyroid tissue from the indicated genotypes. N = 3 mice per genotype. **** P < 0.0001 versus PTH-Cre. C) qRT-PCR for ARC in liver tissue from the indicated genotypes. Each bar in panel C represents mRNA samples isolated from a single mouse.</p

Generalized deletion of ARC in Pdx1-Cre; Men1 f/f mice does not significantly change islet tumor load.

<p>A) Body weights (both genders). B) Fasting plasma insulin concentrations (both genders). C) Fasting blood glucose concentrations (both genders). D) and E) Fasting insulin and glucose measurements in females. F) and G) Fasting insulin and glucose measurements in males. * P < 0.05 Pdx1-Cre; Men1 f/f; ARC -/- versus Pdx1-Cre; Men1 f/f; ARC +/+.</p