blca cell lines j82  (ATCC)

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: Clinical characteristics of patients with bladder cancer (BlCa) *

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques:

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. Bar plot of the expression of a subset of the investigated T-UCRs (48 of 293) with expression increases greater than 2 fold and expression decreases lower than −2.3 fold in BlCa and normal bladder epithelium (NBE) tissues. B. Bar plot of the expression of a subset of the investigated T-UCRs (48 of 141) with expression increases greater than 1 fold and expression decreases lower than −1.66 fold in BlCa and pericancerous BlCa (PBlCa) tissues. C. Comparison of the fold change in expression of 50 T-UCRs for which two different controls (NBE and PBlCa tissues) were used. The outlying ultraconserved RNA (uc).8+ is shown in red. D. RNA was extracted from 18 BlCa and adjacent PBlCa tissues. Evaluation of uc.8+ expression was assessed by quantitative real-time polymerase chain reaction (qRT-PCR). The expression of uc.8+ is higher in PBlCa than in BlCa tissues. ***P<0.001. E. Box plot of the fold change in uc.8+, uc.195+, uc.339+, and uc.217+A expression in BlCa and NBE samples according to qRT-PCR analysis of at least three biological repeats (subset of 22 BlCa patients and 10 NBE; Table , dataset 4). The bold lines inside the boxes in panels D and E represent the medians. The boxes represent the first (Q1) and the third (Q3) quartiles, and the two whiskers represent the minimum and the maximum values, except for outliers. Circles represent outliers, i.e., values lower than Q1-1.5 (Q3-Q1) or higher than Q3+1.5 (Q3-Q1). P values were obtained using the Mann-Whitney U test. ***P<0.001. F. T-UCR classification with respect to the transcripts as single, multiple, or intergenic is depicted for all T-UCRs and for the group of T-UCRs that are deregulated in BlCa tissues. Selective enrichment of a specific group of T-UCRs was not observed in BlCa tissues. Source data for this figure are available online. Abbreviations: ucRNA, ultraconserved RNA; T-UCR, transcribed ultraconserved region; qRT-PCR, quantitative real-time polymerase chain reaction.

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Expressing, Comparison, Real-time Polymerase Chain Reaction, Quantitative RT-PCR, MANN-WHITNEY

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: Representative images show the expression of uc.8+ by in situ hybridization in A. normal bladder epithelium (NBE), B. low-grade BlCa tissues, and C. high-grade BlCa tissues. A' , B' , and C' represent enlargement of specific areas. D. uc.8+ expression evaluated by qRT-PCR was stratified according to grade of BlCa (n=40; Table , dataset 4). E. uc.8+ expression evaluated by qRT-PCR in BlCa tissues (n=40) was stratified according to stage. The bold lines inside the boxes in panels D and E represent the medians. The boxes represent the first (Q1) and the third (Q3) quartiles, and the two whiskers represent the minimum and the maximum values, except for outliers. Circles represent outliers, i.e., values lower than Q1-1.5 (Q3-Q1) or higher than Q3+1.5 (Q3-Q1). P values were obtained using the Mann-Whitney U test. ***P<0.001.

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Expressing, In Situ Hybridization, Quantitative RT-PCR, MANN-WHITNEY

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. Schematic representation of the intronic localization of uc.8+ within CASZ1 . CASZ1 exons are indicated by black boxes. The locations of the uc.8+ forward (F) and reverse (R) primers used for qRT-PCR and the probe used for in situ hybridization are shown. Of the siRNAs targeting CASZ1 , siRNA-1 is located at the 5′ untranslated region (UTR), siRNA-2 is located in exon 6, and siRNA-3 is located at the 3′ UTR. B. RNA levels of CASZ1 and uc.8+ were determined by qRT-PCR in BlCa (n=19, black dots) and control normal bladder epithelium (NBE) samples (n=11, empty circles). Results are presented as means ± standard deviation (SD). Spearman correlation coefficient and P values are indicated. C. CASZ1 expression after silencing of uc.8+. The expression of CASZ1 was not affected in J82 cells transfected with three different siRNAs anti-uc.8+ or siRNA control. D. J82 cells were transfected with siRNA anti- CASZ1 or siRNA control. The CASZ1 level was determined by qRT-PCR. uc.8+ expression was not affected by any of the siRNAs anti- CASZ1 used. Data are expressed as the mean ± SD of triplicate values.

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Quantitative RT-PCR, In Situ Hybridization, Control, Standard Deviation, Expressing, Transfection

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. Representation of the genomic localization of CASZ1 with respect to 1p36.22 (red) obtained using the UCSC Genome Browser (University of California Santa Cruz). B. Representation of the seven T-UCRs in CASZ1 according to their genomic locations with respect to protein-coding genes ( CASZ1 defined using the RefSeq database). C. The expression levels for all T-UCRs located in CASZ1 were measured using qRT-PCR. RNA was extracted from BlCa tissues obtained from 24 patients (dark gray) and 17 normal bladder epithelium (NBE) samples (gray) (Table , Dataset 1). The bold lines inside the boxes represent the medians. The boxes represent the first (Q1) and the third (Q3) quartiles, and the two whiskers represent the minimum and the maximum values, except for outliers. Circles represent outliers, i.e., values lower than Q1-1.5 (Q3-Q1) or higher than Q3+1.5 (Q3-Q1). D. Representative negative correlation of CASZ1 and T-UCR (uc.2+–uc.7+) expression in patients with BlCa (n=14). qRT-PCR analysis results of CASZ1 expression (abscissa) versus T-UCR expression are shown.

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Expressing, Quantitative RT-PCR

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. Schematic representation of the transcript including uc.8+ with respect to CASZ1 . J82 RNA was retrotranscribed by using the SMARTer Rapid Amplification of cDNA Ends (RACE) cDNA Amplification kit (Clontech). Primers used for the 5′ RACE were as follows: Universal Primer Mix (UPM) that recognized the SMARTer oligonucleotide added at the 5′ end, gene specific primers 1 (GSP1) that recognized the sense transcript, and GSP2 primers that recognized the antisense transcript. The arrows represent the direction of amplification from the gene-specific primers that successfully amplified the unknown regions of the TUC8 gene. B. 5′- and 3′-RACE polymerase chain reaction (PCR) performed to amplify the uc.8+ cDNA. C. Sequence of the complete uc.8+ transcript (2435 bases) as determined using RACE. The yellow sequence was reported by Bejerano et al , 2004 [v1].

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Rapid Amplification of cDNA Ends, Amplification, Polymerase Chain Reaction, Sequencing

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. J82 cells were transfected with siRNA anti-uc.8+ or siRNA control and were seeded in 96-well plates. Cell proliferation was determined at the indicated time points. The number of cells per well was measured by the absorbance at 595 nm. The results show data from at least three independent experiments. Cell growth after transfection with siRNA-3 anti-uc.8+ was not significantly different from that of cells transfected with siRNA-2 anti-uc.8+. P values were obtained using the Student t test for independent samples. *P<0.05. B. J82 cells were transfected with siRNA-3 anti-uc.8+ or siRNA control for 72 h, and analysis of cell-cycle distribution was performed by flow cytofluorometry. Bars represent the means and standard deviation (SD) of three experiments. P values were obtained using the Student t test for independent samples. *P<0.05. C. J82 cells were transfected with siRNA-3 anti-uc.8+ or siRNA control. After 24 h, a single scratch was made in the urothelial monolayer. Cell migration was quantified by measuring the distance between the invading front of the cells in three randomly selected microscopic fields (magnification 20x) for each condition and time point. The degree of motility is expressed as the percentage of wound closure compared with the zero time point. Data are representative of three experiments and are expressed as means ± SD. P values were obtained using the Student t test for independent samples. **P<0.01 and ***P<0.001. D. Representative views of wound healing assay was captured and recorded at 0 and 36 h demonstrating reduced migration of J82 cells after uc.8+ silencing. The scale bar in the image is 300 μm. Magnification, 100× (panel I–VI). E. The number of invasive J82 cells in mock, scrambled siRNA, and siRNA-3 anti-uc.8+ groups, which were significantly higher than those in the siRNA-3 anti-uc.8+–transfected group. Data are representative of three experiments and are expressed as means ± SD. P values were obtained using the Student t test for independent samples. **P<0.01 and ***P<0.001. F. Representative microscopic images (magnification, 20×) with crystal violet staining of migrated J82 cells. Mock, scrambled siRNA, and siRNA-3 anti-uc.8+ transfected cells are shown (panel I to III).

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Transfection, Control, Standard Deviation, Migration, Wound Healing Assay, Staining

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. Representative positive correlation between the expression of uc.8+ and that of miR-596 in BlCa samples from 20 patients (Table , dataset 4) measured using qRT-PCR. Correlation was computed using the Spearman correlation coefficient. B. Expression of miR-596 in J82 cell extracts after retrieval of endogenous uc.8+ with a peptide nucleic acid (PNA)/uc.8+ probe. Data are expressed as the means ± standard deviation (SD) of triplicate values. P values were obtained using the Student t test for independent samples. ***P<0.001. C. Relative expression of miR-596 in siRNA anti-uc.8+–transfected J82 cells. Data are expressed as the means ± SD of triplicate values. P values were obtained using the Student t test for independent samples. ***P<0.001. D. J82 cells were transfected with a PNA mimic of miR-596 (PNA-596). Minimum free energy = −51.10 kcal/mol. Data are expressed as the means ± SD of triplicate values. P values were obtained using the Student t test for independent samples. ***P<0.001. E. Predicted co-folded secondary structure of uc.8+ (green sequence) bound to miR-596 (in red), according to the RNAfold browser. F. The blue sequence shows that PNA-596 is perfectly complementary to uc.8+.

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Expressing, Quantitative RT-PCR, Standard Deviation, Transfection, Sequencing

Journal: Oncotarget

Article Title: Long non-coding RNA containing ultraconserved genomic region 8 promotes bladder cancer tumorigenesis

doi: 10.18632/oncotarget.7833

Figure Lengend Snippet: A. Images acquired using inverted fluorescence microscope (magnification, 20×) of J82 control cells (Mock) after transfection with PNA-TO scramble-R8 (PNA-scramble) or with TO-PNA1-R8, the PNA complementary to uc.8+ (PNA-uc.8+). Images were recorded with excitation wavelength (lex)=450–490 nm (DAPI) or lex=510–540 nm (PNA-TO); the superimposition of the images recorded is also reported (Merge). All images were taken with the same confocal microscopy settings. Scale bar, 100 mm. Nuclei of J82 cells were stained with DAPI (blue). B. Inverse correlation between the expression of microRNA (miR)-596 and MMP9 in BlCa samples from 20 patients measured using qRT-PCR. C. Relative expression of MMP9 in siRNA-3 anti-uc.8+ –transfected J82 cells. Endogenous uc.8+ levels in the control cells are shown in grey. Data are expressed as the means ± standard deviation of triplicate values. P values were obtained using the Student t test for independent samples. **P<0.01.

Article Snippet: Human BlCa cell lines J82 (ATCC) and RT112 (European Collection of Cell Cultures) were cultured as a monolayer in Minimal Essential Medium and Roswell Park Memorial Institute medium, respectively, supplemented with 10% fetal bovine serum.

Techniques: Fluorescence, Microscopy, Control, Transfection, Confocal Microscopy, Staining, Expressing, Quantitative RT-PCR, Standard Deviation