Journal: The Journal of Biological Chemistry

Article Title: Identification of Phosphomethylethanolamine N -Methyltransferase from Arabidopsis and Its Role in Choline and Phospholipid Metabolism *

doi: 10.1074/jbc.M110.112151

Figure Lengend Snippet: PtdCho synthesis in plants and yeast highlighting the complementation strategy used to identify the gene encoding PMEAMT. A, the methylation of PEA is a committing step in plant PtdCho synthesis with subsequent methylations at the P-base or Ptd-base level. PEAMT catalyzes the methylation of all three P-bases (heavy arrows) leading to PCho synthesis, whereas PMEAMT cannot use PEA as a substrate. B, yeast synthesizes PtdCho by the Ptd-base route (dashed arrows) that is defective in S. cerevisiae strain CPBY19. Provision of MEA in the medium allows for rescue of PtdCho production in this strain through a by-pass afforded by Arabidopsis PMEAMT.

Article Snippet: When added to SD medium, the ethanolamine (EA), methylethanolamine (MEA), or choline was supplemented at 1 m m . Cloning PMEAMT by Heterologous Complementation The Saccharomyces cerevisiae yeast mutant CPBY19 ( ura3-52 leu2 Δ 1 his3 Δ 200 trp Δ 63 opi3 :: HIS3 cho2 :: LEU2 ) ( 18 ) was transformed using a lithium acetate method ( 21 ) with purified plasmid DNA prepared from the Arabidopsis (Landsberg erecta ecotype) whole seedling cDNA library available in the yeast expression vector pFL61 (ATCC catalog no. 77500) ( 22 ).

Techniques: Methylation

Journal: The Journal of Biological Chemistry

Article Title: Identification of Phosphomethylethanolamine N -Methyltransferase from Arabidopsis and Its Role in Choline and Phospholipid Metabolism *

doi: 10.1074/jbc.M110.112151

Figure Lengend Snippet: Heterologous complementation of PtdCho synthesis in S. cerevisiae CPBY19 by Arabidopsis cDNAs encoding P-base methyltransferases. The CPBY19 (cho2, opi3) mutant strain was grown on SD medium supplemented with 1 mm EA (left) or 1 mm MEA (right). Yeast was untransformed (1) or transformed with pFL61carrying cDNA encoding either AtPMEAMT (2) or AtPEAMT (3).

Article Snippet: When added to SD medium, the ethanolamine (EA), methylethanolamine (MEA), or choline was supplemented at 1 m m . Cloning PMEAMT by Heterologous Complementation The Saccharomyces cerevisiae yeast mutant CPBY19 ( ura3-52 leu2 Δ 1 his3 Δ 200 trp Δ 63 opi3 :: HIS3 cho2 :: LEU2 ) ( 18 ) was transformed using a lithium acetate method ( 21 ) with purified plasmid DNA prepared from the Arabidopsis (Landsberg erecta ecotype) whole seedling cDNA library available in the yeast expression vector pFL61 (ATCC catalog no. 77500) ( 22 ).

Techniques: Mutagenesis, Transformation Assay

Journal: Journal of Clinical Investigation

Article Title: BVES regulates EMT in human corneal and colon cancer cells and is silenced via promoter methylation in human colorectal carcinoma

doi: 10.1172/jci44228

Figure Lengend Snippet: Figure 1 BVES expression is significantly downregulated, and it is mislocalized in human colon carcinoma. (A) A human BVES hydrolysis probe was used to perform qPCR on an Origene cDNA array (TissueScan Cancer Survey I). Ct values were normalized to β-actin and then presented as fold reduction from matched normal (N) (n = 3 per tissue type) and tumor (T) tissue (n = 9 per tissue type). ***P < 0.001, 2-tailed unpaired t test. (B) Analysis of the combined Moffit Cancer Center and Vanderbilt Medical Center colon tumor expression array data set (10, normal samples; 6, adenomas; 33, stage I; 76, stage 2; 82, stage 3; and 59, stage 4; for combined total of 250 CRC samples). For whisker plots, the bottom and top of the boxes are the 25th and 75th percentile, respectively (the lower and upper quartiles, respectively), and the band near the middle of the box is the 50th percentile (median). The whiskers extend to the most extreme data points, which are no more than 1.5 times the interquartile range from the box. *P = 0.04, ***P = 0.001, ****P = 0.0001. (C) Representative immunofluorescence images from normal colon or adenocarci- noma (original magnification, ×400) of H&E, BVES (green), ZO-1 (red), DAPI (blue), and merged images.

Article Snippet: BVES TaqMan qRT-PCR was performed on a multicancer tissue cDNA array (TissueScan Cancer Survey I, OriGene) according to the manufacturers protocol.

Techniques: Expressing, Whisker Assay, Immunofluorescence

Journal: Oncotarget

Article Title: SETD6 regulates NF-κB signaling in urothelial cell survival: Implications for bladder cancer

doi: 10.18632/oncotarget.14750

Figure Lengend Snippet: ( A ) Validation of relative SETD6 expression in different bladder cells was carried out using qPCR. p ≤ 0.05 were considered to be statistically significant (*). The samples were measured in triplicates and the experiment was repeated 3 times. ( B ) cDNA array was performed with bladder cancer tissues ( n = 24) representing different stages and SETD6 expression was analyzed by qPCR. ( C ) Basal level of SETD6 protein was detected using anti-SETD6 antibody by western blotting. ( D ) Immunocytochemistry in different bladder cancer cell lines cells showing cytoplasmic and nuclear SETD6. ( E ) SETD6 protein was detected using anti-SETD6 antibody by western blot in bladder cancer tissues ( n = 9) compared to non-cancerous bladder tissues.

Article Snippet: cDNA array (Origene, TissueScanTM Cancer and Normal Tissue cDNA Arrays) was used to determine the level of SETD6 expression in different bladder tissue samples ( n = 24).

Techniques: Biomarker Discovery, Expressing, Western Blot, Immunocytochemistry