Journal: Molecular Cancer Research

Article Title: Downregulation of Filamin A Interacting Protein 1-Like is Associated with Promoter Methylation and Induces an Invasive Phenotype in Ovarian Cancer

doi: 10.1158/1541-7786.mcr-11-0162

Figure Lengend Snippet: Figure 2. Inverse correlation of FILIP1L expression with the invasive potential of ovarian cell lines. A, Matrigel cell invasion assay for IOSE and ovarian cancer cell lines. Matrigel invasion was measured by using the BD BioCoat Tumor Invasion System as described in Materials and Methods. The y-axis represents a percent change over serum-free control. Error bars indicate SEM (n ¼ 4). P value comparison between each cell line and IOSE523 were: NS for OVCAR8 and OV90; P < 0.0001 for SKOV3, OCC1 and ES2; P ¼ 0.0028 for OVCA429. The result is representative of 2 independent experiments. B, a significant inverse correlation of the FILIP1L mRNA expression with invasiveness of the cells (P ¼ 0.0123 by Spearman's rank correlation method). y-axis; invasiveness of the cells of a percent change over serum-free control shown in (A) was used. x-axis; standardized FILIP1L mRNA expression shown in Figure 1A was used. C, immunoblot analysis for FILIP1L in ES2 and SKOV3 cells transfected with either control or FILIP1LDC103 cDNA. GAPDH blot is shown as the loading control. The result is representative of 3 independent experiments. D, Matrigel cell invasion assay for the same cells used in (C). Transfected cells were subject to invasion assay at 24 hours after transfection. The same experimental procedures were followed as described in (A). P value comparison was: P < 0.0001 for ES2 cells; P ¼ 0.0025 for SKOV3 cells. The result is representative of 3 independent experiments. Representative image of an area scan is shown in Supplementary Figure S2.

Article Snippet: Human ovarian cancer cell lines including ES2, SKOV3, and OV90 were purchased from the American Type Culture Collection.

Techniques: Expressing, Invasion Assay, Control, Comparison, Western Blot, Transfection

Journal: Molecular Cancer Research

Article Title: Downregulation of Filamin A Interacting Protein 1-Like is Associated with Promoter Methylation and Induces an Invasive Phenotype in Ovarian Cancer

doi: 10.1158/1541-7786.mcr-11-0162

Figure Lengend Snippet: Figure 5. Association of reduced methylation in the FILIP1L promoter with restoration of FILIP1L expression in ovarian cancer cells following treatment with a DNA demethylating agent. A, real-time RT-PCR analysis for FILIP1L on cDNA from ES2, OVCA429, SKOV3, and OVCAR8 cells treated with either 5-aza-20-DAC or TSA. The y-axis represents a fold change over DMSO-treated control cells where each value was standardized with the housekeeping gene hRPL7. Error bars indicate SEM (n ¼ 3). P value comparison between DMSO-treated control and either DAC- or TSA-treated experiments were: both 0.1 and 1 mmol/L DAC (P < 0.0001) and both 0.1 and 1 mmol/L TSA (not significant; NS) for ES2; both 10 and 25 mmol/L DAC (P < 0.0001) and both 0.3 and 1 mmol/L TSA (NS) for OVCA429; 1 mmol/L DAC (P ¼ 0.0002), 3 mmol/L DAC (P ¼ 0.0004), and both 0.3 and 1 mmol/L TSA (NS) for SKOV3; 0.1 mmol/L DAC, 1 mmol/L DAC, and 0.1 mmol/L TSA (NS), and 1 mmol/L TSA (P ¼ 0.0003) for OVCAR8. The result is an average of 3 independent experiments. B, immunoblot analysis for FILIP1L in the same cells used in (A). GAPDH blot is shown as the loading control. Note that in FILIP1L-high expressing OVCAR8 cells, DAC treatment did not result in increased FILIP1L expression, whereas TSA treatment decreased FILIP1L expression. The result is representative of 3 independent experiments. C, DNA methylation status of the CpG island in the FILIP1L promoter from the same cells used in (A) was analyzed by Sequenom EpiTYPER Mass Array. Mass Array results are shown as described in Figure 4B. The result is an average of 3 independent experiments. D, Mass Array results are shown as described in Figure 4C from the same cells used in (A). P value comparison between DMSO-treated control and DAC- or TSA-treated experiments were: 1 mmol/L DAC (P < 0.0001) and 1 mmol/L TSA (NS) for ES2; 25 mmol/L DAC (P ¼ 0.0009) for OVCA429; 3 mmol/L DAC (P < 0.0001) for SKOV3; 1 mmol/L DAC (NS) for OVCAR8. Note that either TSA-treated ES2 or DAC-treated OVCAR8 did not decrease DNA methylation compared with DMSO-treated control.

Article Snippet: Human ovarian cancer cell lines including ES2, SKOV3, and OV90 were purchased from the American Type Culture Collection.

Techniques: Methylation, Expressing, Quantitative RT-PCR, Control, Comparison, Western Blot, DNA Methylation Assay