rt112 bladder cancer cells  (Institut Curie)

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG1 and STAG2 show both overlapping and unique distributions over genomic elements and chromatin states in RT112 cells. ( A ) ChIP-Seq read density heatmaps for STAG1, STAG2, and SMC1 at common, STAG1-enriched (STAG1 > STAG2), and STAG2-enriched (STAG2 > STAG1) cohesin positions within a peak-centered 6kb window. ( B ) Read density distribution for STAG1 and STAG2 at common, STAG1-enriched, and STAG2-enriched positions within a peak-centered 6kb window. ( C ) Bar-plot diagram showing the distribution of common, STAG1-enriched, and STAG2-enriched cohesin positions over genomic elements. ( D ) Distribution of cohesin-bound genomic sites throughout chromatin states identified in RT112 cells by ChromHMM and based on combinations of histone modifications and CTCF (see for definition of chromatin states). ( E ) Peak-centered enrichment plot for CTCF over the three categories of cohesin-bound positions showing relative depletion in STAG2-enriched sites.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: ChIP-sequencing

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG2 loss in RT112 cells does not interfere with A/B compartments or TAD boundaries. ( A ) Western blot analysis of control (shNT) and STAG2-silenced RT112 cells showing efficient depletion of STAG2 at the protein level. ( B ) Reproducibility, measured by a stratum-adjusted correlation coefficient (SCC), between pair-wise comparisons of Hi-C datasets. ( C ) Hi-C matrices for chr2 at 500 kb resolution in cells transduced with control or STAG2-targeting shRNAs. The darker red reflects a greater frequency of interaction. ( D ) Compartment tracks for chr2 at 100 kb resolution as determined by the values of the first principal component (PC1) in control and STAG2-silenced cells. ( E ) Expression, as defined by RNA-Seq (log 2 FPKM), of genes within compartments A and B. As expected, genes assigned to compartment A are more transcriptionally active than genes in compartment B. t -test: *** P < 0.001. ( F ) Compartmentalization saddle plots: average intra-chromosomal interaction frequencies between 200kb bins, normalized by expected interaction frequency based on genomic distance. Bins are sorted by their PC1 value derived from control cells Hi-C data. Preferential B-B interactions are in the upper left corner, and preferential A-A interactions are in the lower right corner. Numbers in corners represent the strength of AA interactions as compared to AB interactions and BB interactions over BA interactions. ( G ) Scatterplot of PC1 values of the eigenvectors of intrachromosomal interaction matrices for control and STAG2-silenced cells. The Venn diagrams show the overlap in terms of compartment-switching bins between sh1 and sh2. The number of genes mapped to genomic bins switching compartments is also indicated. Only one GO term is significantly enriched (FDR < 0.01) among genes switching from A to B: GO:0050907 (detection of chemical stimulus involved in sensory perception). ( H ) Effect of STAG2-depletion on the number of TADs per chromosome. Boxplot notches represent the confidence interval around the median. The number of total TADs is indicated below the boxplots. ( I ) Histograms depicting the strength of the TAD borders detected in control and STAG2-silenced cells, according to the TADbit score. ( J ) Average insulation profile around TAD boundaries (±600 kb) in control and STAG2-silenced cells. ( K ) Density plot depicting the distribution of TAD sizes identified in control and STAG2-silenced cells. ( L ) Hi-C normalized interaction matrices for chr2 at 100kb resolution comparing TAD organization in control and STAG2-silenced cells. ( M ) Effect of STAG2-depletion on conservation of TAD borders. Boxplot notches represent the confidence interval around the median.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: Western Blot, Control, Hi-C, Transduction, Expressing, RNA Sequencing, Derivative Assay, Insulation

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG2 depletion leads to deregulation of the basal/luminal transcriptional programs in RT112. ( A ) Scatter plots of expression values (FPKM) of genes in control versus STAG2-silenced cells. Statistically significant differentially expressed genes are highlighted in dark (FDR < 0.05) or light red ( P < 0.05). ( B ) Scatter plot showing a positive and significant correlation between gene expression changes in sh1 and sh2 (left). Venn diagrams displaying the overlap between sh1 and sh2 in terms of significant up- and down-regulated genes. ( C ) GSEA enrichment plots of gene sets associated with the luminal and basal subtypes of muscle-invasive UBC showing significant deregulation in STAG2-silenced RT112 cells. ( D ) Distribution of STAG2 expression (FPKM) in the UROMOL cohort of 476 UBC samples , highlighting the thresholds of the first and fourth quartiles (119 samples per group). We defined ‘STAG2 high’ cases as those with expression values in the fourth quartile, and ‘STAG2 low’ cases as those with STAG2 levels in the first quartile. ( E ) GSEA enrichment plots for genes down-regulated in STAG2-silenced cells in ‘STAG2 high’ versus ‘STAG2 low’ tumor samples. ( F , G ) Heatmaps displaying relative expression values (Z-score of FPKM) of genes significantly down-regulated in RT112 cells with sh1 (F) or sh2 (G) and in ‘STAG2 low’ versus ‘STAG2 high’ tumor samples.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: Expressing, Control, Gene Expression

Journal: Nucleic Acids Research

Article Title: STAG2 loss-of-function affects short-range genomic contacts and modulates the basal-luminal transcriptional program of bladder cancer cells

doi: 10.1093/nar/gkab864

Figure Lengend Snippet: STAG2 silencing is accompanied by rewiring of DNA contacts. ( A ) MD plots depicting the fold change in interaction frequency of DNA contacts in control (shNT) versus STAG2-silenced cells in relation to the linear distance between interacting regions for chromosome 15. Interactions showing statistically significant differences (adjusted P < 0.05) are highlighted in red (gained) or blue (lost). ( B ) Scatter plot comparing changes in interaction frequency between cells transduced with sh1 versus cells transduced with sh2. Gained and lost interactions are highlighted in red and blue, respectively. ( C ) Interaction frequency of lost and gained contacts in shNT and STAG2-silenced cells. ( D ) Distance between peaks of lost and gained DNA contacts. Gained interactions span longer distances than lost interactions. ( E ) Top four scoring motifs enriched in the subsets of interactions defined in C. For a more extensive list of significantly enriched motifs, see . ( F ) Distribution of control, lost, and gained interactions over compartments and ( G ) chromatin states in RT112 cells (see ). ( H ) Left: chromatin contact network generated from the 20kb resolution Hi-C interaction map of control cells, showing in pink the nodes involved in contacts that are lost upon STAG2 silencing. Right: chromatin assortativity of nodes that lose contacts as the network is filtered eliminating contacts spanning short distances.

Article Snippet: RT112 bladder cancer cells used at CNIO and Institut Curie were from the same original stock; HEK293T cells (transformed human embryonic kidney) were from the ATCC.

Techniques: Control, Transduction, Generated, Hi-C

Journal: BMC Biology

Article Title: Association of Bacteroides acidifaciens relative abundance with high-fibre diet-associated radiosensitisation

doi: 10.1186/s12915-020-00836-x

Figure Lengend Snippet: Soluble high fibre causes increased growth delay in irradiated bladder cancer cell xenografts, and responses are influenced by gut microbiota composition. a Tumour growth in RT112 flank xenografts irradiated with 6 Gy IR, in mice fed low-fibre, high mixed fibre, high insoluble fibre, and high soluble fibre diets ( n = 8 for each group). Tumour curve slopes were calculated by linear regression to represent tumour growth rates and compared by ANOVA. b The Kaplan-Meier survival curves for mice showing plots of time to treble tumour volume. c Mice in the soluble HF group were stratified into responders and non-responders based on tumour radiation response. d Shannon’s index of gut microbiota in responders and non-responders by the Kruskal-Wallis test. Error bars represent the interquartile range of diversity scores. e Principal coordinate analysis of gut samples ( n = 8) in the soluble HF group by response using the Bray-Curtis dissimilarity

Article Snippet: At 7 to 8 weeks of age, mice were injected subcutaneously with RT112 bladder cancer cells (DSMZ, Germany) and started receiving either a low-fibre diet (2 g cellulose/3850 kcal), a high insoluble fibre diet (100 g cellulose/3850 kcal), a high soluble fibre diet (100 g inulin/3850 kcal), or a high mixed fibre diet (50 g cellulose + 50 g inulin/3850 kcal) for a maximum time of 9 weeks or until they were culled when the tumours reached 350 mm 3 .

Techniques: Irradiation

Journal: BMC Biology

Article Title: Association of Bacteroides acidifaciens relative abundance with high-fibre diet-associated radiosensitisation

doi: 10.1186/s12915-020-00836-x

Figure Lengend Snippet: Differences in composition of the gut microbiome between responders and non-responders. a Taxonomic cladogram from LEfSe showing differences among taxa between responders and non-responders in the soluble HF group. Dot size is proportional to the abundance of the taxon. b Linear discriminant analysis (LDA) scores computed for differentially abundant taxa in the microbiomes of responders (green) and non-responders (red). Length indicates the effect size associated with a taxon, p = 0.05 for the Kruskal-Wallis test. c Discrete false-discovery rate of different abundant taxa in responders and non-responders in the soluble HF group. Differential abundance within all taxonomic levels in responders versus non-responders by Mann-Whitney U test. Dots are overlapping between Bacteroides acidifaciens and Allobaculum , and between Lactobacillus and Parabacteroides . Relative abundance of d B. acidifaciens and e Bacteroidales S24-7 and in responders and non-responders in the soluble HF group. f , g Metagenomic functional prediction by PICRUSt of the gut microbiome in responders ( n = 4) and non-responders ( n = 4) in the soluble HF group with reference to the KEGG database level 2. Columns represent mice (responders, orange; non-responders, blue), and rows represent enrichment of predicted KEGG pathways (red, low enrichment; yellow, medium enrichment; blue, high enrichment). h Western blot analysis of histone acetylation levels of RT112 cells treated with SCFAs ( N = 3). i Linear quadratic survival curves of IC10-treated RT112 cells with receiving irradiation of 0–8 Gy ( N = 3). j Cell survival analysis of RT112 cells treated with single SCFA and combined SCFAs mixture ( N = 3). Combined (purple bar) denotes SCFA mixture of 10 mM acetate, 5 mM propionate, and 1.7 mM butyrate. k Reduced cell survival of RT112 cells by bacterial supernatants at day 2 ( N = 1). BA+FP denotes the cross-feeding of B. acidifaciens and F. prausnitzii , while Bif+FP denotes the cross-feeding of Bifidobacterium and F. prausnitzii. * p < 0.05; ** p < 0.01; *** p < 0.001

Article Snippet: At 7 to 8 weeks of age, mice were injected subcutaneously with RT112 bladder cancer cells (DSMZ, Germany) and started receiving either a low-fibre diet (2 g cellulose/3850 kcal), a high insoluble fibre diet (100 g cellulose/3850 kcal), a high soluble fibre diet (100 g inulin/3850 kcal), or a high mixed fibre diet (50 g cellulose + 50 g inulin/3850 kcal) for a maximum time of 9 weeks or until they were culled when the tumours reached 350 mm 3 .

Techniques: MANN-WHITNEY, Functional Assay, Western Blot, Irradiation

Journal: Oncology Letters

Article Title: MKP-1 overexpression is associated with chemoresistance in bladder cancer via the MAPK pathway

doi: 10.3892/ol.2020.11741

Figure Lengend Snippet: MKP-1 and MAPK expression in RT112 cells transfected with NC and MKP-1 siRNA. (A) Relative MKP-1 expression in the siNC and siMKP-1 groups was examined using reverse transcription-quantitative PCR. **P<0.01. (B) Representative microscopic images of siNC and siMKP-1 treated cells captured in both 2D and 3D environments under a phase contrast microscope. (C) MKP-1 expression of the siNC and siMKP-1 groups, as determined via western blotting. GAPDH was used as the internal control. (D) Phosphorylated and total ERK1/2 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (E) Phosphorylated and total p38 protein expressions of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. (F) Phosphorylated and total JNK protein expression levels of the siNC and siMKP-1 group, as determined via western blotting. GAPDH was used as the internal control. MKP-1, mitogen activated protein kinase phosphatase-1; NC, negative control; siMKP-1, MKP-1 small interfering RNA; siNC, small interfering negative control.

Article Snippet: The human bladder cancer cell line RT112 was purchased from the Leibniz Institute DSMZ and maintained in RPMI 1640 medium supplemented with 10% fetal bovine serum at 37°C in a humidified atmosphere containing of 5% CO 2 .

Techniques: Expressing, Transfection, Reverse Transcription, Real-time Polymerase Chain Reaction, Microscopy, Western Blot, Control, Negative Control, Small Interfering RNA