The characteristics of patients with bladder cancer.

<p>The characteristics of patients with bladder cancer.</p

Mass spectrometry analysis of the proteins pull-down by GAS5.

<p>Mass spectrometry analysis of the proteins pull-down by GAS5.</p

GAS5 negatively regulates CDK6 expression.

<p>(A) Biotinylated GAS5 or control were incubated with nuclear extracts (RT4 cells), targeted with streptavidin beads and associated proteins were resolved in a gel. Silver staining of the SDS-PAGE gel containing aliquots of samples derived from proteins pulled down by GAS5. (B) RIP experiments were performed using CDK6 antibody to immunoprecipitate RNA and a primer to detect GAS5 RNA. (C) Analysis of CDK6 mRNA level was performed in bladder cancer cell lines after GAS5-siRNA treatment. (D) Western blot analysis of CDK6 protein level was performed in bladder cancer cells treated with GAS5-siRNA. (E) Analysis of CDK6 mRNA level was performed in bladder cancer cell lines after GAS5 overexpression. The results show data from at least three independent experiments, expressed as the mean ± SD. *<i>P</i><<i>0.05</i>. (F) Negative correlation between GAS5 levels and the CDK6 levels in 16 bladder cancer samples (<i>r</i>² = 0.168, <i>p = 0.013</i>).</p

GAS5 inhibits bladder tumor cell proliferation.

<p>(A) RT4 cells were treated with GAS5-siRNA, and GAS5 expression level was assayed after 48 h by real-time PCR. (B) RT4 cells were treated with GAS5-siRNA, and at the indicated time points, the numbers of cells per well were measured by the absorbance (450 nm) of reduced WST-8. (C) RT4 cells were treated with pcDNA-GAS5, and GAS5 expression level was assayed by real-time PCR. (D) RT4 cells were transiently overexpressed with GAS5, and the numbers of cells per well were measured by the absorbance (450 nm) of reduced WST-8. The results show data from at least three independent experiments, expressed as the mean ± SD. *<i>P</i><<i>0.05</i>.</p

GAS5 regulates bladder cancer cell cycle by regulating CDK6.

<p>(A) GAS5 expression was inhibited by specific siRNAs in RT4, and the cell apoptosis was analyzed by flow cytometer 48 h later. (B) RT4 cells were treated with GAS5-siRNA or CDK6-siRNA. Forty-eight hours later, the relative cell numbers in each cell cycle phase after propidium iodide staining were determined by FACS analysis. The data are from one of three independent experiments. The histograms were analyzed and the percentage of cells in each phase of the cell cycle is shown. The results are presented as mean ± SD for three experiments.</p

Induction of BTCC cell arrest at cell cycle G2-M phase following ROC1 knockdown.

<p>A and B, ROC1 knockdown induced G2-M cell cycle arrest in 253J (A) and 5637 (B) cells. The cells were transfected with siROC1 or siCONT for 48–120h, and cell cycle profile was detected using FACS analyses following Propidium Iodide (PI) staining. C and D, ROC1 knockdown induced G2 cell cycle arrest. 253J (C) and 5637 (D) cells were transfected with siROC1 or siCONT for 96h, and subjected to FACS analysis after phospho-histone 3(PH3)/PI double staining. E and F, Expression of cell cycle-associated protein in 253J (E) and 5637 (F) cells were examined using western blot post-transfection at the indicated time interval. Representative results of three independent experiments are shown. Columns, mean of three independent experiments; bars, SEM. ** <i>P</i><0.01.</p

ROC1 knockdown-induced bladder cancer cell growth inhibition was partially rescued by suppression of p21 expression.

<p>253J and 5637 cells were transfected with the indicated siRNA for 96h, and subjected to western blot analyses (A and B), CCK8 assay (C and D), senescence analyses with SA-β-gal staining (E), and FACS analysis with PI staining (F). Representative results of three independent experiments are shown. Columns, mean of three independent experiments; bars, SEM. ** <i>P</i><0.01 and *** <i>P</i><0.001.</p

ROC1 knockdown inhibited the growth of bladder cancer cell 5637 xenografts <i>in vivo.</i>

<p>A, Representative photographs of tumors isolated from nude mice in each group 7 weeks following injection of LTR and LTC. B, IHC staining of xenografts tissues with the indicated antibody in both groups. C and D, Tumor growth curve (C) and tumor weight (D) in both groups. E, ROC1 and p21 protein in extracted protein from xenografts in both groups were determined using western blot analyses. At the end of the experiments, tumor tissues were excised from each mouse and weighed. Columns, mean weight of 5 tumors from individual mice in each group; bars, SEM. * <i>P</i><0.05, ** <i>P</i><0.01 and *** <i>P</i><0.001.</p