Journal: PLoS Genetics

Article Title: Hypomethylation of a LINE-1 Promoter Activates an Alternate Transcript of the MET Oncogene in Bladders with Cancer

doi: 10.1371/journal.pgen.1000917

Figure Lengend Snippet: (A) Map of alternate transcript from L1- MET . Exons are represented by black boxes and a red box represents the specific L1. The bent arrows indicate transcriptional start sites and ATGs indicate translational start sites. Horizontal arrows indicate the primers for PCR of bisulfite converted DNA and RT–PCR. The bisulfite-specific primers Bi-L1-5′ and Bi-MET-3′ were used to amplify L1- MET for methylation analysis and Bi-L1-5′ and Bi-L1-3′ for global L1 methylation analysis. The RT–PCR primers, RT-L1-MET-5′ and RT-MET-3′ were used to amplify cDNA of the L1- MET transcript for expression analysis and RT-MET-3′ and RT-MET-5′ for the full length MET expression analysis. The lower tick marks represent each CpG site. Vertical arrows indicate the CpG sites analyzed by the Ms-SNuPE assay. (B) L1- MET methylation (red bars) and L1 methylation (black bars) was analyzed by Ms-SNuPE in 8 normal tissues, one normal bladder fibroblast cell line (LD419), two non-tumorigenic urothelial cell lines (UROtsa and NK2426), and 20 bladder carcinoma cell lines. Values are the average of one CpG site for L1 and an average of two CpG sites for L1- MET from technical triplicates. Error bars represent the standard deviation. (C) Expression of L1- MET was measured using real-time RT PCR in one normal bladder fibroblast cell line, two normal urothelial cell lines and 10 bladder carcinoma cell lines. There is clearly a strong correlation between DNA methylation and expression in all 13 cell lines examined. Values are the average from technical duplicates. Red bars indicate the methylation status of L1- MET , which is also represented in (B), and green bars represent the level of expression relative to GAPDH .

Article Snippet: Human bladder carcinoma cell lines were obtained commercially (T24, J82, HT1376, SCaBER, UM-UC-3, TCCSUP, and RT4; American Type Culture Collection, Manassas, VA) or derived in our laboratory (prefix LD).

Techniques: Reverse Transcription Polymerase Chain Reaction, Methylation, Expressing, Snupe Assay, Standard Deviation, Quantitative RT-PCR, DNA Methylation Assay

Journal: PLoS Genetics

Article Title: Hypomethylation of a LINE-1 Promoter Activates an Alternate Transcript of the MET Oncogene in Bladders with Cancer

doi: 10.1371/journal.pgen.1000917

Figure Lengend Snippet: (A) DNA methylation at L1- MET and global L1s was determined by pyrosequencing in the immortalized urothelial cell line UROtsa and bladder carcinoma cell line T24. Chromatin immunoprecipitation was performed using antibodies for H3K4me3, acetylated H3, and H2A.Z. The values of the ChIP assay are the average of three experiments with technical duplicates. Error bars represent the standard deviation, and p16 represents a single copy gene control. The presence of active histone marks was associated with absence of DNA methylation at L1- MET in the cancer cell line. Methylase dependent single promoter analysis (MSPA) with M. CviP I, a GpC methyltransferase, of the (B) endogenously methylated L1- MET promoter (ch7:116364020–116364664) in the UROtsa immortalized urothelial cell line and the (C) endogenously unmethylated L1- MET promoter in T24 bladder carcinoma cells. (D) DNA methylation at L1- MET and global L1s was determined by pyrosequencing in the colon cancer cell line HCT116 and HCT116 DKO cells (DNMT1 hypomorph/DNMT3B knockout) , . Chromatin immunoprecipitation was performed using antibodies for H2A.Z. The presence of active histone marks was associated with absence of DNA methylation at L1- MET in the DKO cell line. Methylase dependent single promoter analysis (MSPA) with M. CviP I, a GpC methyltransferase, of the (E) endogenously methylated L1- MET promoter in HCT116 colon cancer cells, and (F) endogenously unmethylated L1- MET promoter in HCT116 DKO cells. White circles indicate unmethylated sites and black circles indicate methylated sites. Orange bars indicate areas of protection consistent with the presence of a nucleosome.

Article Snippet: Human bladder carcinoma cell lines were obtained commercially (T24, J82, HT1376, SCaBER, UM-UC-3, TCCSUP, and RT4; American Type Culture Collection, Manassas, VA) or derived in our laboratory (prefix LD).

Techniques: DNA Methylation Assay, Chromatin Immunoprecipitation, Standard Deviation, Control, Methylation, Knock-Out

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibitory activities of Taiwanese local pomegranate fruit. The ethylacetate, butanol, and water layers extracted from pomegranate peel extract (PEP) were tested for the inhibitory efficacy to T24 ( A ) or J82 ( B ) cells using MTT assay as described in Materials and Methods. PEPE2 and PEPE3 ( C ) were also investigated for the inhibitory effectiveness to T24 or J82 cells. 0.1% ( v / v ) Dimethyl sulfoxide (DMSO)-treated urinary bladder urothelial carcinoma (UBUC) cells were regarded as the solvent control. Each MTT result was the typical data of at least three independent experiments. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: MTT Assay, Solvent, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The propidium iodide (PI) and PI/annexin V analyses of UBUC cells treated with PEPE2. The results of PI analyses were represented in ( A ) the T24 cells and ( B ) the J82 cells. The data of the PI/annexin V measurement were shown in ( C ) T24 cells and ( D ) J82 cells. Each flow cytometry figure was the typical result of three independent experiments. The diagram under the PI/annexin V panel was the results of the three independent experiments. The cell cycles and apoptosis detection of the T24 or J82 cells treated with PEPE2 were measured with PI and PI/annexin V analyses, respectively, using flow cytometry as described in the . The 0.1% ( v / v ) DMSO-treated UBUC cells were implemented as the vehicle control. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Flow Cytometry, Control

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The molecular mechanisms of apoptotic pathway evoked in PEPE2-incubated UBUC cells. T24 and J82 cells were treated with 50 and 20 μg/mL PEPE2 respectively. Then the protein levels of ( A ) pro-/cleaved caspase-3; ( B ) pro-/cleaved caspase-8, DR4 and DR5; ( C ) pro-/cleaved caspase-9, Bax and Bcl-2 and ( D ) Bip, VCP and pro- caspase-12 in PEPE2-treated T24 cells were measured using western immunoblotting as described in the supporting information. The 0.1% ( v / v ) DMSO-treated UBUC cells were used as the solvent control; ( E ) The proposed molecular apoptotic pathway provoked in the PEPE2-treated UBUC cells. The immunoblot in each figure was the representative result of at least three independent experiments. The diagram (ratio (mean ± standard deviation (S.D.)) under each immunoblot indicated the ratio of the normalized protein intensity (observed protein/actin) of PEPE2-treated cells at the indicated time interval, divided by that at the 0-h time point. * p ≤ 0.05, ** p ≤ 0.01, *** p ≤ 0.001.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Incubation, Western Blot, Solvent, Control, Standard Deviation

Journal: Nutrients

Article Title: Deciphering the Molecular Mechanism Underlying the Inhibitory Efficacy of Taiwanese Local Pomegranate Peels against Urinary Bladder Urothelial Carcinoma

doi: 10.3390/nu10050543

Figure Lengend Snippet: The inhibition of the xenografted UBUC growth in nude mice by the treatment of the EtOAc layer. T24 cells were injected s.c. into nude mice and were o.g. administrated with water (control), 2, 5, 10, or 100 mg/kg EtOAc layer as described in Materials and Methods. ( A ) The body weights of the extract-treated mice. *** p < 0.001, ** p < 0.005; ( B ) The effects of the EtOAc layer on tumor growth in the xenografted nude mice. The volumes of tumors from the extract-fed mice were compared to those of the water-fed mice. *** p < 0.001; ( C ) The tumor weight. The tumor weight was measured at the 10th week. *** p < 0.001; ( D ) The typical H/E images (400×) of tumor specimens dissected from xenografted nude mice; ( E ) The representative terminal deoxynucleotidyl transferase-mediated deoxyuridine triphosphate nick end-labeling (TUNEL) images (400×) of tumor lesions from xenografted nude mice. H&E and TUNEL assays were performed as described in Material and Methods.

Article Snippet: Human UBUC J82 cells recognized as high grade were provided by Dr. Chien-Feng Li from Department of Pathology, Chi-Mei Medical Center, Tainan, Taiwan and maintained at 37 °C in Dulbecco’s Modified Eagle Medium supplemented with 10% ( v / v ) fetal bovine serum.

Techniques: Inhibition, Injection, Control, End Labeling, TUNEL Assay