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1.
FIGURE 2.

FIGURE 2. From: Epigenetic Control of the Invasion-promoting MT1-MMP/MMP-2/TIMP-2 Axis in Cancer Cells.

MCF-7 cells synthesize TIMP-2 but not MT1-MMP and MMP-2. A, Western blot analysis of cell surface-associated, biotin-labeled MT1-MMP (top panel) and gelatin zymography of secretory MMP-2 (bottom panel). U-MT/PDX cells which produce both the proenzyme and the enzyme of MT1-MMP were used as a reference in Western blots. B, Western blot analysis of secretory and cellular TIMP-2 (top and middle panels, respectively). rTIMP-2 was used as a reference. Bottom panel, Western blotting of cellular α-actin (loading control).

Andrei V. Chernov, et al. J Biol Chem. 2009 May 8;284(19):12727-12734.
2.
FIGURE 1.

FIGURE 1. From: Epigenetic Control of the Invasion-promoting MT1-MMP/MMP-2/TIMP-2 Axis in Cancer Cells.

Migration efficiency of cells. A, cells (1 × 104) were allowed to migrate for 12 h in the wells of a Transwell plate using a membrane coated with type I collagen. B, an inhibitor of MMPs (GM6001) inhibits migration of HT1080 and U-MT/PDX cells. Where indicated, GM6001 (10 μm) was added to the cells. Migration efficiency was expressed as a percentage of the cells, which have migrated through the membrane and which were detected on the membrane undersurface relative to the total number of cells. The data are from three independent experiments performed in triplicate.

Andrei V. Chernov, et al. J Biol Chem. 2009 May 8;284(19):12727-12734.
3.
FIGURE 3.

FIGURE 3. From: Epigenetic Control of the Invasion-promoting MT1-MMP/MMP-2/TIMP-2 Axis in Cancer Cells.

MCF-7 cells do not express MT1-MMP and MMP-2 mRNA. A, an RT-PCR analysis of the expression of the MT1-MMP, MMP-2, and TIMP-2 genes in TP98G, U373, U251, U87, and MCF-7 cells. The primers we used are shown in supplemental Table S1. The specific fragments of the expected size (193, 292, and 183 bp for MT1-MMP, MMP-2, and TIMP-2, respectively) were amplified. Glyceraldehyde-3-phosphate dehydrogenase (GAPDH) was used as a loading control. B, methylation-sensitive PCR of the MT1-MMP and MMP-2 promoter regions genes in TP98G, U373, U251, U87, and MCF-7 cells. UM and M, the PCR products are generated using the primers specific for unmethylated and methylated DNA, respectively. C, methylation-sensitive PCR of analysis of the MT1-MMP and MMP-2 promoter regions in MCF-7 cells stably transfected with the full-length MT1-MMP gene (+MT1-MMP) and the full-length β3 integrin subunit (+integrin). The bottom panel shows the results of the analysis of untreated MCF-7 cells and MCF-7 cells subjected to demethylation using 5-aza-2′-deoxycytidine (+Aza-dC).

Andrei V. Chernov, et al. J Biol Chem. 2009 May 8;284(19):12727-12734.
4.
FIGURE 6.

FIGURE 6. From: Epigenetic Control of the Invasion-promoting MT1-MMP/MMP-2/TIMP-2 Axis in Cancer Cells.

Epigenetic analysis of the TIMP-2 gene in cancer cells. A, schematic presentation of the human TIMP-2 promoter region. The first exon is shown as an open box. The arrow shows the transcription start site. The gray boxes indicate the two CpG-rich regions, which are localized 1.7-kb and 0.5-kb upstream of the transcription start site. Black boxes show the regions which were analyzed using the bisulfite sequencing (BIS1 and BIS2). B, the bisulfite sequencing of the 329-bp-long BIS1 fragment with 16 CpG islands and the 220-bp-long BIS2 fragment with 12 CpG islands in TP98G, U373, U251, U87, and MCF-7 cells. The methylation status of each CpG dinucleotide was determined by sequencing of at least six independent PCR clones of bisulfite-converted DNA. The primers we used are shown in supplemental Table S3. Each circle represents an individual CpG. Open and closed circles indicate unmethylated and methylated CpGs, respectively.

Andrei V. Chernov, et al. J Biol Chem. 2009 May 8;284(19):12727-12734.
5.
FIGURE 5.

FIGURE 5. From: Epigenetic Control of the Invasion-promoting MT1-MMP/MMP-2/TIMP-2 Axis in Cancer Cells.

Epigenetic analysis of the MMP-2 gene in cancer cells. A, schematic presentation of the human MMP-2 promoter region. The first exon is shown as an open box. The arrow shows the transcription start site. The gray boxes indicate the two CpG-rich regions. Black boxes show the regions that were analyzed using the bisulfite sequencing (BIS1 and BIS2) and MSP. The B1–B5 regions were analyzed using ChIP. B, bisulfite sequencing of the 221-bp-long BIS1 fragment with 22 CpG islands and the 230-bp-long BIS2 fragment with 6 CpG islands in U251 and MCF-7 cells. The methylation status of each CpG dinucleotide was determined by sequencing at least six independent PCR clones of bisulfite-converted DNA. The primers we used are shown in supplemental Table S3. Each circle represents an individual CpG. Open and closed circles indicate unmethylated and methylated CpGs, respectively. C and D, ChIP analysis of the MMP-2 promoter region in U251 and MCF-7 cells, respectively. Gray bars, the Ct values of the transcriptionally active RLP30 control gene. Open bars, the Ct values which correspond to the B1-B5 primer pairs in the order shown in A. H3ac, H3K4me2, H3K27me3, and H3K4me3 represent the analyzed histone modifications. Total H3 histone ChIP was used as an additional control.

Andrei V. Chernov, et al. J Biol Chem. 2009 May 8;284(19):12727-12734.
6.
FIGURE 4.

FIGURE 4. From: Epigenetic Control of the Invasion-promoting MT1-MMP/MMP-2/TIMP-2 Axis in Cancer Cells.

Epigenetic analysis of the MT1-MMP gene in cancer cells. A, schematic presentation of the human MT1-MMP promoter region. The upstream L52 gene is on the left. The first exon is shown as an open box. The arrows show the transcription start sites. The gray box indicates the 1.4-kb-long CpG island. Black boxes show the regions that were analyzed using the bisulfite sequencing (BIS) and MSP. The A1–A6 regions were analyzed using ChIP. B, the bisulfite sequencing of the 240-bp-long BIS fragment containing 15 CpG islands in TP98G, U373, U251, U87, and MCF-7 cells. The methylation status of each CpG dinucleotide was determined by sequencing of at least seven independent PCR clones of bisulfite-converted DNA. The primers we used are shown in supplemental Table S3. Each circle represents an individual CpG. Open and closed circles indicate unmethylated and methylated CpGs, respectively. C and D, ChIP analysis of the MT1-MMP promoter region in U251 and MCF-7 cells, respectively. Gray bars, the Ct values of the transcriptionally active RLP30 control gene. Open bars, the Ct values which correspond to the A1–A6 primer pairs in the order shown in A. H3ac, H3K4me2, H3K27me3, and H3K4me3 represent the analyzed histone modifications. Total H3 histone ChIP was used as an additional control.

Andrei V. Chernov, et al. J Biol Chem. 2009 May 8;284(19):12727-12734.

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