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: Frontiers in Microbiology

Article Title: Testosterone increases the virulence traits of uropathogenic Escherichia coli

doi: 10.3389/fmicb.2024.1422747

Figure Lengend Snippet: CFT073 colonization ( A,C 4 h) or invasion ( B , 2 h) of bladder epithelial cells after CFT073 pre-treated with or without testosterone (100 pg/mL, 2 ng/mL and 60 ng/mL). CFT073 (carrying a GFP-expressing plasmid) colonization was measured as mean fluorescence intensity (MFI) (A) and imaged (C) . Data are presented as mean ± SD of n = 3–8 independent experiments. The asterisk distinguishes statistical significance: * = p < 0.05, ** = p < 0.01, and *** = p < 0.001 vs. CFT073. Scale bar: 500 μm.

Article Snippet: Human bladder epithelial cell line 5,637 was purchased from American Type Culture Collection (Manassas, VA, United States) and cultured with Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM non-essential amino acids (all from Thermo Fisher Scientific) at 37°C with 5% CO 2 .

Techniques: Expressing, Plasmid Preparation, Fluorescence

Journal: Frontiers in Microbiology

Article Title: Testosterone increases the virulence traits of uropathogenic Escherichia coli

doi: 10.3389/fmicb.2024.1422747

Figure Lengend Snippet: IL-1β (A) , IL-8 (B) and LDH (C) release from bladder epithelial cells stimulated with CFT073 MOI10 pre-treated with or without testosterone (100 pg/mL, 2 ng/mL and 60 ng/mL) at 4 h. Data are presented as mean ± SD of n = 6–8 independent experiments. The asterisks distinguish statistical significance: * = p < 0.05 and **** = p < 0.0001.

Article Snippet: Human bladder epithelial cell line 5,637 was purchased from American Type Culture Collection (Manassas, VA, United States) and cultured with Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM non-essential amino acids (all from Thermo Fisher Scientific) at 37°C with 5% CO 2 .

Techniques:

Journal: Frontiers in Microbiology

Article Title: Testosterone increases the virulence traits of uropathogenic Escherichia coli

doi: 10.3389/fmicb.2024.1422747

Figure Lengend Snippet: RNASE7 (A) , LL37 (B) , beta-defensin 1 (C) and beta-defensin 2 (D) release from bladder epithelial cells stimulated with CFT073 MOI10 pre-treated with or without testosterone (100 pg/mL, 2 ng/mL and 60 ng/mL) at 4 h. Data are presented as mean ± SD of n = 3 independent experiments. The asterisks distinguish statistical significance: * = p < 0.05 and **** = p < 0.0001.

Article Snippet: Human bladder epithelial cell line 5,637 was purchased from American Type Culture Collection (Manassas, VA, United States) and cultured with Dulbecco’s Modified Eagle Medium (DMEM) (Lonza, Basel, Switzerland) supplemented with 10% fetal bovine serum (FBS), 2 mM L-glutamine and 1 mM non-essential amino acids (all from Thermo Fisher Scientific) at 37°C with 5% CO 2 .

Techniques:

Journal: Cancer Science

Article Title: Cancer‐testis antigen BORIS is a novel prognostic marker for patients with esophageal cancer

doi: 10.1111/j.1349-7006.2012.02355.x

Figure Lengend Snippet: Expression of BORIS m RNA in various cancer cell lines and cancer tissues and presence of BORIS ‐specific IgG in sera from patients with various cancers

Article Snippet: The cell lines used in the study were esophageal squamous cell carcinoma cell lines, TE2, TE3, TE4, TE5, TE6, TE7, TE8, TE9, TE10, TE11, TE12, TE13, TE14, and TE15 (Tohoku University, Sendai, Japan); melanoma cell lines, SKmel23, SKmel28, 888mel, A375mel, 1363mel, 928mel, 624mel, 501Amel, 586mel, 526mel, 501mel, 397mel, and 1362mel (Surgery Branch, NCI, NIH, Bethesda, MD, USA); colon cancer cell line, COLO205 (JCRB, Osaka, Japan); breast cancer cell line HS578 (American Type Culture Collection (ATCC), Manassas, VA, USA); stomach cancer cell lines, MKN1, MKN7, MKN28, MKN46, and MKN74 (Yamagata University, Yamagata, Japan); endometrial cancer cell line SNGII (Keio University, Tokyo, Japan); prostate cancer cell line LNCaP (ATCC); bladder cancer cell line, KU7 (Keio University); and brain tumor cell line U87MG (ATCC).

Techniques: Expressing

Journal: EMBO Molecular Medicine

Article Title: Phosphorylation of TFCP2L1 by CDK1 is required for stem cell pluripotency and bladder carcinogenesis

doi: 10.15252/emmm.201910880

Figure Lengend Snippet: A Western blot analysis of protein expression in primary normal human bladder epithelial cells (HBlEpC) and two human BC cell lines (J82 and T24). B–E Cell proliferation of HBlEpC (B), T24 (C), and basal and luminal subtypes of MIBC and NMIBC cell lines (E) after infection with lentiviruses containing human TFCP2L1 or CDK1 ORFs or TFCP2L1 shRNA (two independent shRNAs; #1 and #2). (D) Ectopic expression or silencing of TFCP2L1 and CDK1 in T24 cells was validated by Western blot analysis. F Tumor sphere formation in T24 cells after TFCP2L1 silencing, ectopic expression of TFCP2L1, or TFCP2L1 and CDK1 co‐expression. Images are shown at ×40 (upper panel) or ×100 (lower panel) magnification. Scale bars = 200 μm. G Tumor sphere formation assay in basal and luminal subtypes of MIBC and NMIBC cell lines with ectopic expression or silencing of TFCP2L1 . The representative images for each cell line are available as Fig A and B. H Clonogenic limiting dilution assay of T24 cells with ectopic expression of TFCP2L1 or CDK1 and TFCP2L1 . I Matrigel invasion assays with the indicated T24 cells. Representative images are shown at ×200 magnification. Scale bars = 100 μm. Data information: All quantitative data are mean ± SEM. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with cells transfected with the empty control vector, # P < 0.05, ## P < 0.01, n.s. = not significant. Statistical tests used are as follows: one‐way (H, I) and two‐way ANOVA (B, C, E, F, G) with Bonferroni post hoc tests. Number of biological replicates is n ≥ 4. The exact P ‐values and number of replicates can be found in the source data. Source data are available online for this figure.

Article Snippet: In addition, human primary epithelial cells derived from normal human bladder (HBlEpC; Cell Applications, Inc, San Diego, CA) and human BC cell lines J82, T24, 5637, HT1197, HT1376, and RT4 (purchased from ATCC, Manassas, VA) were employed.

Techniques: Western Blot, Expressing, Infection, shRNA, Tube Formation Assay, Limiting Dilution Assay, Transfection, Control, Plasmid Preparation