Journal: Cancer Research Communications

Article Title: STAG2 Expression is Associated with Adverse Survival Outcomes and Regulates Cell Phenotype in Muscle-invasive Bladder Cancer

doi: 10.1158/2767-9764.CRC-22-0155

Figure Lengend Snippet: STAG2 loss augments cisplatin treatment in MIBC. A, Kaplan–Meier OS (left) and PFS (right) survival of MIBC TMA patient samples treated with cisplatin, stratified into STAG2-low (H-score 0–50, n = 12) and -high (H-score 50–300, n = 20) groups. P = 0.125 for OS, P = 0.036 for PFS; P value computed using log-rank test. B, Scanned images of crystal violet-stained HB-CLS-1, TCC-SUP, T24, and BO2 colonies after 24 hours of treatment and 14 days of growth. Left: control, shSTAG2-1, and shSTAG-2 clonal populations treated with vehicle. Right: populations treated with 0.1 μg/mL (TCC-SUP, HB-CLS-1), 0.25 μg/mL (BO2) or 0.5 μg/mL (T24) cisplatin. Images are representative of three individual experiments. C, Surviving fraction of cells relative to untreated control for HB-CLS-1, TCC-SUP, T24, and BO2 cell lines with and without cisplatin treatment. P values determined by unpaired Student t test. Comparisons made between untreated shSTAG2-1/2 and untreated controls, and shSTAG2-1/2+CDDP to control+CDDP. Data represented as mean + SEM. CDDP: cisplatin. Individual comparisons made using unpaired Student t test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Article Snippet: We utilized four cell lines with STAG2 KD via shSTAG2-1/2 (HB-CLS-1, TCC-SUP, T24, and BO2) in addition to UM-UC-3 cells with STAG2 overexpression.

Techniques: Staining, Control

Journal: Oncology Letters

Article Title: Silencing of ECHDC1 inhibits growth of gemcitabine-resistant bladder cancer cells

doi: 10.3892/ol.2017.7269

Figure Lengend Snippet: Analysis of gemcitabine resistance of the bladder cancer cell lines. Bladder cancer cells were treated with gemcitabine for 72 h and cell viability was analyzed using CCK-8. The viability of parental UMUC3 (A) and HT1376 (B) cells was respectively suppressed compared to (A) UMUC3GR and (B) HT1376GR by treatment with gemcitabine in a dose-dependent manner. Values are expressed as means ± standard deviation.

Article Snippet: Cell culture The human bladder cancer cell lines, UMUC3 and HT1376, which were used in this study, were purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: CCK-8 Assay, Standard Deviation

Journal: Oncology Letters

Article Title: Silencing of ECHDC1 inhibits growth of gemcitabine-resistant bladder cancer cells

doi: 10.3892/ol.2017.7269

Figure Lengend Snippet: Proteins with increased expression in gemcitabine-resistant cells identified using iTRAQ proteomic analysis.

Article Snippet: Cell culture The human bladder cancer cell lines, UMUC3 and HT1376, which were used in this study, were purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Expressing, Multiplex sample analysis, Histone Deacetylase Assay

Journal: Oncology Letters

Article Title: Silencing of ECHDC1 inhibits growth of gemcitabine-resistant bladder cancer cells

doi: 10.3892/ol.2017.7269

Figure Lengend Snippet: ECHDC1 protein expression in gemcitabine resistant and sensitive cell lines. (A) ECHDC1 protein expression levels in parental and gemcitabine resistant (GR) cell lines were determined using western blotting. ECHDC1 expression levels were clearly increased in the gemcitabine-resistant cells compared to the gemcitabine-sensitive cells. (B) Immunofluorescence analysis of ECHDC1 expression (red) in the indicated cells. Nuclei were counterstained with Hoechst 33342 (blue). The red signal in the cytoplasm, reflecting ECHDC1 expression, was much stronger in UMUC3GR than in UMUC3 cells.

Article Snippet: Cell culture The human bladder cancer cell lines, UMUC3 and HT1376, which were used in this study, were purchased from DS Pharma Biomedical (Osaka, Japan).

Techniques: Expressing, Western Blot, Immunofluorescence

Journal: bioRxiv

Article Title: circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

doi: 10.1101/2023.09.14.557527

Figure Lengend Snippet: (A) circHIPK3 is produced from the second exon of the HIPK3 gene. We designed a circHIPK3-specific siRNA targeting the backsplice junction of circHIPK3 to specifically knockdown the expression of circHIPK3. (B) Expression of circHIPK3 and the corresponding linear transcript in eleven bladder cancer cell lines. UMUC3 and J82 were chosen for knockdown experiments based on circHIPK3 expression levels and cell line stability. CPM = counts per million. (C) Knockdown efficiency of circHIPK3 in UMUC3 and J82 at different time points post circHIPK3 (yellow) or scramble (grey) siRNA transfection. Expression is normalized to GAPDH levels. (D) PCA plot of gene expression in UMUC3 and J82. PCA plots are based on the genes with the 50% most variance across all cell line samples. Gene expression is log transformed (natural logarithm) and added a pseudocount of 1. (E) Overlap of differentially expressed genes across time and/or conditions between UMUC3 (n = 3,072) and J82 (n = 2,389). P-value obtained by Fisher’s Exact Test. (F ) Number of differentially expressed genes at each time point (Wald test, Benjamini-Hochberg correction with FDR < 0.1). Only genes with perturbed expression profiles across time and/or conditions in UMUC3 (n = 3,072) or J82 (n = 2,389) are considered. (G) Differential expression analysis between circHIPK3 KD and scr siRNA samples 24 hour post-transfection (Wald test). The log2 fold changes (circHIPK3 KD vs scr) are plotted against the negative log10(p-values). Colors indicate if genes are significantly down- (red) or upregulated (blue) or not differentially expressed (Not DE, green) after Benjamini-Hochberg correction, FDR < 0.1. Vertical lines indicate a log2FC > 1 or < −1.

Article Snippet: The overlap was largest for downregulated genes in both UMUC3 (P < 2e-15, Chi-square test, ) and J82 cells (P < 0.001, Chi-square test, Supplementary Figure S3H).

Techniques: Produced, Knockdown, Expressing, Transfection, Gene Expression, Transformation Assay, Quantitative Proteomics

Journal: bioRxiv

Article Title: circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

doi: 10.1101/2023.09.14.557527

Figure Lengend Snippet: (A) Illustration of conserved miRNA binding sites in circHIPK3 based on AGO-CLIP data. (B) Percentage of genes in each group with miR-148ab-3p/152 target sites (CAGUGCA) in their 3’UTRs. Gene regulation is based on circHIPK3 KD vs scr siRNA transfection 24 hours (24h) post-transfection in J82. P-values obtained by Chi-square Test. (C) Procedure for motif enrichment analysis using Regmex. First, we extract all 7-mers in the sequence of circHIPK3. For each 7-mer, e.g., CCCTGCC, we identify their presence in the 3’UTR of genes. We order all genes according to a differential expression score calculated as the −log10(P-value) multiplied by the fold change direction, e.g., 1 for upregulated genes and −1 for downregulated genes. Then we calculate a motif score for each 7-mer based on their occurrences in either down- or upregulated genes. If a 7-mer has a positive motif score it means that it is enriched in the 3’UTR of genes that are upregulated upon circHIPK3 KD. Conversely, 7-mers with a negative motif score are primarily found in genes that are downregulated upon circHIPK3 KD. The 7-mer, CCCTGCC, is used for illustration purposes. (D) Regmex motif scores for circHIPK3 7-mers (UMUC3, 24h). Alignment of the ten 7-mers with the most negative motif scores are shown. 7-mers are ranked from most negative to most positive motif scores. Numbers correspond to rank. (E) Illustration of circHIPK3 and position of the ten motifs with the lowest motif scores. The 7-mers with the most negative motif scores found in circHIPK3 comprise a larger 11-mer, AGGCCCCCAGC, present in the sequence of circHIPK3. (F) Percentage of genes in each group containing the 11-mer motif upon circHIPK3 KD in UMUC3 cells (24h). P-values obtained by Chi-square Test.

Article Snippet: The overlap was largest for downregulated genes in both UMUC3 (P < 2e-15, Chi-square test, ) and J82 cells (P < 0.001, Chi-square test, Supplementary Figure S3H).

Techniques: Binding Assay, Transfection, Sequencing, Quantitative Proteomics

Journal: bioRxiv

Article Title: circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

doi: 10.1101/2023.09.14.557527

Figure Lengend Snippet: (A) Illustration of RBP binding sites in circHIPK3 based on eCLIP data in the ENCODE cell lines HepG2 and K562. (B) RNA immunoprecipitation of circHIPK3-RBPs and others in HepG2 and K562 cells. Bands confirm that circHIPK3 interacts with IGF2BP1 and IGF2BP2. Marker indicates 50 bp. (C) Percentage of genes in each group containing IGF2BP2 binding sites (K562) in UMUC3 (24h). P-values obtained by Chi-square Test. (D) Overlap between genes that are affected by circHIPK3 KD in UMUC3 and IGF2BP2 KD in K562. P-value obtained by Fisher’s Exact Test. (E) Percentage of genes in each group affected by IGF2BP2 KD in K562. P-values obtained by Chi-square Test. (F) Regulation of genes affected by both circHIPK3 KD (UMUC3, 24h) and IGF2BP2 KD (K562). The x-axis indicates gene regulation upon circHIPK3 KD in UMUC3. Percentage on y-axis and colors show how these genes are regulated upon IGF2BP2 KD. Downregulated genes upon circHIPK3 KD are mainly upregulated upon IGF2BP2 KD and vice versa. (G) Regmex motif enrichment analysis upon IGF2BP2 KD in K562. 7-mers are ranked from most positive to most negative motif scores. The ten 7-mers with the most negative motif scores in UMUC3 are shown. Numbers indicate motif rank. All possible 7-mers are evaluated (n = 16,384). (H-I) Percentage of genes in each group containing the 11-mer motif (H) and IGF2BP2 binding sites (I) upon IGF2BP2 KD in K562. P-values obtained by Chi-square Test.

Article Snippet: The overlap was largest for downregulated genes in both UMUC3 (P < 2e-15, Chi-square test, ) and J82 cells (P < 0.001, Chi-square test, Supplementary Figure S3H).

Techniques: Binding Assay, RNA Immunoprecipitation, Marker

Journal: bioRxiv

Article Title: circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

doi: 10.1101/2023.09.14.557527

Figure Lengend Snippet: (A) Relative enrichment of circHIPK3 levels between IP and input for the three RBPs GRWD1, IGF2BP1, and IGF2BP2 in the bladder cancer cell line FL3. GFP was used as a negative control. (B) Relative enrichment of circHIPK3 levels between IP and input for IGF2BP2 upon circHIPK3 overexpression (OE). GFP was used as a negative control. Error bars reflect standard deviation of biological triplicates. (C) Percentage of genes in each group containing the 11-mer motif upon IGF2BP2 KD in UMUC3. P-values obtained by Chi-square Test. (D) Gene set enrichment analysis of 50 hallmarks of cancer upon circHIPK3 KD (24h) in UMUC3 cells (FDR < 0.1 for all shown pathways). (E) Downregulated hallmarks of cancer upon IGF2BP2 KD in UMUC3 (P-value < 0.05 for all shown pathways).

Article Snippet: The overlap was largest for downregulated genes in both UMUC3 (P < 2e-15, Chi-square test, ) and J82 cells (P < 0.001, Chi-square test, Supplementary Figure S3H).

Techniques: Negative Control, Over Expression, Standard Deviation

Journal: bioRxiv

Article Title: circHIPK3 nucleates IGF2BP2 and functions as a competing endogenous RNA

doi: 10.1101/2023.09.14.557527

Figure Lengend Snippet: (A) STAT3 mRNA expression upon circHIPK3 KD in the time-course perturbation experiment in UMUC3 cells. Expression represents DESeq2 normalized counts. P-value (24h) obtained by Wald Test. (B) Expression of STAT3 upon IGF2BP2 knockdown in UMUC3 cells. P-value obtained by Wald Test. (C) Relative enrichment of STAT3 and β-actin levels between IGF2BP2 IP and input in FL3 cells. GFP was used as a negative control. P-value obtained by T-test. (D) Relative enrichment of STAT3 levels between IGF2BP2 IP and input upon circHIPK3 KD in FL3 cells. P-value obtained by T-test. Control sample (scr) has been normalized to 1. (E) Relative enrichment of STAT3 and GAPDH levels between IGF2BP2 IP and input after 11-mer ASO transfection in FL3 cells. P-values obtained by T-test. (F) Relative enrichment of circHIPK3, STAT3 , and GAPDH levels between IGF2BP2 IP and input after transfection of wildtype and mutated circHIPK3 in HEK293 cells. P-values obtained by T-test. (G) TP53 mRNA expression upon circHIPK3 KD in the time-course perturbation experiment in UMUC3 cells. Expression represents DESeq2 normalized counts. P-value (72h) obtained by Wald Test. (H) Western blot of STAT3 and p53 protein upon circHIPK3 KD in UMUC cells. Quantifications on the left are internally normalized to HuR and then scr is normalized to 1. P-values obtained by T-test. (I-J) Immunofluorescence staining for STAT3 (I) or p53 (J) in fixed UMUC3 cells (as indicated - green) subjected to control (scr) or circHIPK3 KD. Nuclei were counterstained using DAPI (as indicated - blue). Mean signal intensities are quantified within nuclei or entire cells to the right. P-values obtained by Wilcoxon Rank Sum Test. (B-F,H) Error bars reflect standard deviation of biological triplicates.

Article Snippet: The overlap was largest for downregulated genes in both UMUC3 (P < 2e-15, Chi-square test, ) and J82 cells (P < 0.001, Chi-square test, Supplementary Figure S3H).

Techniques: Expressing, Knockdown, Negative Control, Control, Transfection, Western Blot, Immunofluorescence, Staining, Standard Deviation

Journal: BMC Cancer

Article Title: Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

doi: 10.1186/1471-2407-14-507

Figure Lengend Snippet: Epithelial-to-mesenchymal markers. A , the endogenous expression levels of the epithelial marker E-cadherin and mesenchymal markers N-cadherin and vimentin were assessed in bladder tumour cells (HT1376, SW780, UMUC3, T24 and 5637) by western blot analysis. B , the effect of 5 μM VFL treatments of 5637 bladder tumour cells for 48 and 72 h on the indicated protein markers was assessed by western blot analysis. C , Cyclin D1 expression levels were assessed by Western blot analysis after treatment of 5637 bladder tumour cells for 48 and 72 h with VFL. Western blot data are representative of three independent experiments and GAPDH antibody was used as loading control for normalization.

Article Snippet: UMUC3 and SW780 were generously donated by Dr. F. Garcia (Pharmamar S.A., Madrid) and 5637 and T24 by Dr. F. Real (Spanish National Cancer Research Centre from Madrid, Spain).

Techniques: Expressing, Marker, Western Blot, Control

Journal: BMC Cancer

Article Title: Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

doi: 10.1186/1471-2407-14-507

Figure Lengend Snippet: Effect of VFL on cytotoxicity of bladder tumour cell lines. The five indicated human tumour bladder cell lines (HT1376, 5637, SW780 in upper panel; UMUC3 and T24 in bottom panel) were treated with increasing concentrations of VFL (0–100 μM) for 48 h. Cell viability was determined by the MTT assay. A , HT1376. B , 5637. C, SW780. D , UMUC3 E , T24. Data are the means ± SEM of three independent experiments represented by logarithmic scale, and the IC50 value and CI95% for each cell line are indicated.

Article Snippet: UMUC3 and SW780 were generously donated by Dr. F. Garcia (Pharmamar S.A., Madrid) and 5637 and T24 by Dr. F. Real (Spanish National Cancer Research Centre from Madrid, Spain).

Techniques: MTT Assay

Journal: BMC Cancer

Article Title: Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

doi: 10.1186/1471-2407-14-507

Figure Lengend Snippet: Effect of VFL on epithelial differentiation and apoptosis. A , Western blot analysis of E-cadherin and Hakai expression levels in HT1373 bladder tumour cells treated with 5 μM VFL for 48 h. B , immunofluorescence analysis of E-cadherin expression in HT1376 cells treated with 5 μM VFL for 48 hours. Scale bar, 200 μM. C , TUNEL staining for the analysis of apoptosis was performed following by immunofluorescence of E-cadherin in HT1376 cells treated with 5 μM VFL for 48 hours, as indicated in material and methods. Scale bar, 50 μM. D , Western blot analysis of the levels of E-cadherin, Hakai, N-cadherin and vimentin in UMUC3 bladder tumour cells treated with 5 μM VFL for 48 hours. Western blots are representative of three independent experiments and GAPDH was assessed as loading control.

Article Snippet: UMUC3 and SW780 were generously donated by Dr. F. Garcia (Pharmamar S.A., Madrid) and 5637 and T24 by Dr. F. Real (Spanish National Cancer Research Centre from Madrid, Spain).

Techniques: Western Blot, Expressing, Immunofluorescence, TUNEL Assay, Staining, Control

Journal: BMC Cancer

Article Title: Role of the microtubule-targeting drug vinflunine on cell-cell adhesions in bladder epithelial tumour cells

doi: 10.1186/1471-2407-14-507

Figure Lengend Snippet: VFL is involved in Hakai degradation via proteasome-mediated in 5637 cell line. A , the levels of Hakai mRNA in 5637 (upper panel, left), HT1376 (upper panel, right), and UMUC3 (lower panel) cells following treatment with 5 μM VFL for 48 hours were determined by RT-qPCR analysis. HPRT mRNA levels were measured for normalization. The means ± SEM are represented from three independent experiments (*p < 0.05, n = 3). B , effect of proteasome inhibitor, MG132, on Hakai expression in 5637. Cells following treatment with 5 μM VFL for 48 hours were incubated for 2 h in the absence or presence of proteasome inhibitor (20 μM MG132) and cell lysates were prepared for Western blot analysis to detect Hakai, E-cadherin and normalization control GAPDH. Western blot data are representative of three independent experiments and quantification by densitometry was represented (lower panel, *p < 0.05, **p < 0.01).

Article Snippet: UMUC3 and SW780 were generously donated by Dr. F. Garcia (Pharmamar S.A., Madrid) and 5637 and T24 by Dr. F. Real (Spanish National Cancer Research Centre from Madrid, Spain).

Techniques: Quantitative RT-PCR, Expressing, Incubation, Western Blot, Control