Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Extracellular ATP promotes proliferation of T24 cells through P2X receptors. ( A ) ATP concentrations in supernatants of T24, RT4, and TRT-HU-1 cell cultures were determined by HPLC (mean ± SD, n = 3 separate experiments; *** p < 0.001, one-way ANOVA). ( B ) T24, TRT-HU-1, and RT4 cells were labeled with CFSE and proliferation was determined at the indicated time points by flow cytometry (mean ± SD, n = 3 separate experiments). ( C ) T24 cells were treated with inhibitors of P2X1 (NF023; 10 µM) and/or P2X7 (A438079; 10 µM) receptors or with the general P2 receptor antagonist suramin (100 µM) and proliferation was assessed at the indicated time points (mean ± SD, n = 3 separate experiments). ( D ) Doubling times of T24 cells treated with P2 receptor inhibitors as in C or with ATP (10 µM), apyrase (1 U/ml), CBX (20 µM), or the P2X4R antagonist 5-BDBD (10 µM) were determined after 72 hours (mean ± SD, n = 3 separate experiments; * p < 0.05, *** p < 0.001 vs. control. # p < 0.05, ## p < 0.01, one-way ANOVA); MFI, mean fluorescence intensity.

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques: Labeling, Flow Cytometry, Fluorescence

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: P2X1, P2X4, and P2X7 receptor expression in muscle-invasive bladder cancer (MIBC). ( A – C ) Representative photomicrographs show TMA samples with low ( A ) and high ( B ) P2X1R expression in cancer cells of MIBC. Compared to smooth muscle cells, muscle infiltrating cancer cells in ( C ) show strong distinct membranous staining. ( D – F ) TMA samples of MIBC with low ( D ) and high ( E ) P2X4R expression are shown. MIBC cells exhibit membranous and partially fine-granular cytoplasmic P2X4R staining, while tumor-infiltrating immune cells show strong immunoreactivity ( F ). ( G – I ) Low and high P2X7R expression patterns in MIBC are shown in ( G ) and ( H ), respectively. Cancer cells between muscle cells and in lymphatic vessels are highlighted by strong membranous P2X7R expression ( I ). Scale bars equal 100 μm (outer pictures and ( C , F , I )) and 20 μm (inserts).

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Demographics, clinicopathological characteristics, and associations with P2X1, P2X4, and P2X7 receptor expression.

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Effect of P2X receptor expression levels on overall survival in muscle-invasive bladder cancer (MIBC). Expression of P2X1R ( A ), P2X4R ( B ), P2X7R ( C ), and combined expression of P2X1R and P2X7R ( D ) was immunohistochemically analyzed in MIBC and scored as “low” (below median) or “high” (above median). Kaplan–Meier overall survival (OS) curves were plotted, and groups were compared by the log-rank test; ( A ) and ( B ): n = 171; ( C ): n = 172; ( D ): n = 170.

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Effect of P2X receptor expression levels on tumor-specific survival (TSS) in muscle-invasive bladder cancer (MIBC). Expression of P2X1R ( A ), P2X4R ( B ), P2X7R ( C ), and combined expression of P2X1R and P2X7R ( D ) was immunohistochemically analyzed in MIBC and scored as “low” (below median) or “high” (above median). Kaplan–Meier tumor-specific survival (TSS) curves were plotted, and groups were compared by the log-rank test; ( A , B ): n = 171; ( C ): n = 172; ( D ): n = 170.

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Effect of P2X receptor expression levels on disease-free survival (DFS) in muscle-invasive bladder cancer (MIBC). Expression of P2X1R ( A ), P2X4R ( B ), P2X7R ( C ), and combined expression of P2X1R and P2X7R ( D ) was immunohistochemically analyzed in MIBC and scored as “low” (below median) or “high” (above median). Kaplan–Meier disease-free survival (DFS) curves were plotted, and groups were compared by the log-rank test; ( A , B ): n = 171; ( C ): n = 172; ( D ): n = 170.

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Multivariate analysis of potential prognostic survival factors in MIBC patients.

Article Snippet: If indicated, antagonists of P2X1R (10 µM NF023; Tocris, Bristol, UK), P2X4R (10 µM 5-BDBD, Tocris), or P2X7R (10 µM A438079, Tocris), the P2 receptor antagonist suramin (100 µM; Sigma-Aldrich), the ATP-degrading enzyme apyrase (1 U/mL; Sigma-Aldrich), the ATP release blocker carbenoxolone (CBX; 20 µM; Sigma-Aldrich), or the natural P2XR ligand ATP (10 µM; Sigma-Aldrich) were added, and the cells were maintained in a final volume of 500 µL in 5% CO 2 /95% air at 37 °C.

Techniques:

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: P2X1, P2X4, and P2X7 receptor expression in muscle-invasive bladder cancer (MIBC). ( A – C ) Representative photomicrographs show TMA samples with low ( A ) and high ( B ) P2X1R expression in cancer cells of MIBC. Compared to smooth muscle cells, muscle infiltrating cancer cells in ( C ) show strong distinct membranous staining. ( D – F ) TMA samples of MIBC with low ( D ) and high ( E ) P2X4R expression are shown. MIBC cells exhibit membranous and partially fine-granular cytoplasmic P2X4R staining, while tumor-infiltrating immune cells show strong immunoreactivity ( F ). ( G – I ) Low and high P2X7R expression patterns in MIBC are shown in ( G ) and ( H ), respectively. Cancer cells between muscle cells and in lymphatic vessels are highlighted by strong membranous P2X7R expression ( I ). Scale bars equal 100 μm (outer pictures and ( C , F , I )) and 20 μm (inserts).

Article Snippet: Slides were incubated with the polyclonal rabbit anti-human P2X1R primary antibody (1:300; Abcam, Berlin, Germany; ab74058) for 60 min at room temperature.

Techniques: Expressing, Staining

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Demographics, clinicopathological characteristics, and associations with P2X1, P2X4, and P2X7 receptor expression.

Article Snippet: Slides were incubated with the polyclonal rabbit anti-human P2X1R primary antibody (1:300; Abcam, Berlin, Germany; ab74058) for 60 min at room temperature.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Effect of P2X receptor expression levels on overall survival in muscle-invasive bladder cancer (MIBC). Expression of P2X1R ( A ), P2X4R ( B ), P2X7R ( C ), and combined expression of P2X1R and P2X7R ( D ) was immunohistochemically analyzed in MIBC and scored as “low” (below median) or “high” (above median). Kaplan–Meier overall survival (OS) curves were plotted, and groups were compared by the log-rank test; ( A ) and ( B ): n = 171; ( C ): n = 172; ( D ): n = 170.

Article Snippet: Slides were incubated with the polyclonal rabbit anti-human P2X1R primary antibody (1:300; Abcam, Berlin, Germany; ab74058) for 60 min at room temperature.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Effect of P2X receptor expression levels on tumor-specific survival (TSS) in muscle-invasive bladder cancer (MIBC). Expression of P2X1R ( A ), P2X4R ( B ), P2X7R ( C ), and combined expression of P2X1R and P2X7R ( D ) was immunohistochemically analyzed in MIBC and scored as “low” (below median) or “high” (above median). Kaplan–Meier tumor-specific survival (TSS) curves were plotted, and groups were compared by the log-rank test; ( A , B ): n = 171; ( C ): n = 172; ( D ): n = 170.

Article Snippet: Slides were incubated with the polyclonal rabbit anti-human P2X1R primary antibody (1:300; Abcam, Berlin, Germany; ab74058) for 60 min at room temperature.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Effect of P2X receptor expression levels on disease-free survival (DFS) in muscle-invasive bladder cancer (MIBC). Expression of P2X1R ( A ), P2X4R ( B ), P2X7R ( C ), and combined expression of P2X1R and P2X7R ( D ) was immunohistochemically analyzed in MIBC and scored as “low” (below median) or “high” (above median). Kaplan–Meier disease-free survival (DFS) curves were plotted, and groups were compared by the log-rank test; ( A , B ): n = 171; ( C ): n = 172; ( D ): n = 170.

Article Snippet: Slides were incubated with the polyclonal rabbit anti-human P2X1R primary antibody (1:300; Abcam, Berlin, Germany; ab74058) for 60 min at room temperature.

Techniques: Expressing

Journal: Cancers

Article Title: P2X1 and P2X7 Receptor Overexpression Is a Negative Predictor of Survival in Muscle-Invasive Bladder Cancer

doi: 10.3390/cancers15082321

Figure Lengend Snippet: Multivariate analysis of potential prognostic survival factors in MIBC patients.

Article Snippet: Slides were incubated with the polyclonal rabbit anti-human P2X1R primary antibody (1:300; Abcam, Berlin, Germany; ab74058) for 60 min at room temperature.

Techniques: