Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: Farnesoid X receptor (FXR) expression and the effects on cell viability in human bladder cancers. ( A , B ) Overall and disease-free survival rate in differential expression of NR1H4 (FXR) gene in TCGA database of bladder cancer patients. ( C ) Scatter plots in differential expression of FXR gene in TCGA database of bladder cancer tissues (red plot) and adjacent normal tissues (blue plot). * p < 0.05 compared with the bladder cancer tissues group. ( D ) The expression levels of FXR and SHP in the bladder cancer cell lines were analyzed by Western blotting. GAPDH was a loading control. * p < 0.05 compared with the T24 group. ( E ) The survival rate of TSGH8301 and T24 were analyzed after doxycycline induced for 72 h in vector control and FXR overexpressed groups using MTT assay. ( F ) Colony formation assay of TSGH8301 and T24 were analyzed after 9 days of doxycycline induction in vector control and FXR overexpressed groups. Wells were visualized and quantified. ** p < 0.01; *** p < 0.001 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Expressing, Quantitative Proteomics, Western Blot, Control, Plasmid Preparation, MTT Assay, Colony Assay

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect on migration and adhesion abilities after FXR overexpression. ( A ) Wound healing migration assays were performed in FXR-overexpressed (FXR-O) TSGH8301 and T24 human bladder cancer cells after 6 h scratch. The right panels display the relative rate of the wound healing migratory ability. ** p < 0.01; *** p < 0.001 compared to the control group. ( B ) Adhesion assays were performed in FXR-O TSGH8301 and T24 cells after 50 min of incubation. Thereafter, the adhered cells were stained and captured. The right panels display the relative rate of the adhesive ability. ** p < 0.01 compared to the control group. Scale bar = 200 μm.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Migration, Over Expression, Relative Rate, Control, Incubation, Staining, Adhesive

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect on focal adhesion complex expression after FXR overexpression. ( A ) Focal adhesion kinase (FAK), phospho-focal adhesion kinase (p-FAK), integrin β1, integrin β3, myosin light chain (MLC) and phospho-myosin light chain (p-MLC) were analyzed by Western blotting in the TSGH8301 and T24 cells after FXR overexpression for 48 and 72 h. GAPDH was used as the loading control. ( B ) The bar graphs show the relative quantitative analysis of the aforementioned proteins. * p < 0.05; ** p < 0.01 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Expressing, Over Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of proteosome inhibitor MG132 and lysosome inhibitor NH4Cl on the migratory and adhesive abilities after FXR overexpression. ( A ) Wound healing migration assays were performed with or without MG132 or NH4Cl in TSGH8301 and T24 cells after 6 h scratch. The right panels displayed quantitative results of the relative migration rate. ** p < 0.01 compared to the control group; ## p < 0.01 compared to the FXR-O group. ( B ) Adhesion assays were performed in TSGH8301 and T24 cells for 50 min. The cells were stained and captured. The right panels displayed quantitative results of the relative adhesive rate. ** p < 0.01 compared to the control group; # p < 0.05 compared to the FXR-O group. Scale bar = 200 μm.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Adhesive, Over Expression, Migration, Control, Staining

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of MG132 and NH4Cl on integrin expression after FXR overexpression. ( A ) Integrin β1, integrin β3, MLC and p-MLC were analyzed by Western blotting in the TSGH8301 and T24 cells with or without MG132 or NH4Cl. GAPDH was used as the loading control. ( B ) The bar graphs show the relative quantitative analysis of the aforementioned proteins. ** p < 0.01 compared with the control group; # p < 0.05 compared to the FXR-O group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Expressing, Over Expression, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of invasive abilities after FXR overexpression. ( A ) Transwell invasion assays were performed for 16 h incubation in the T24 cells after FXR overexpression. The invasive cells were stained and captured. The right panel displayed the quantitative result. ** p < 0.01 compared to the control group. Scale bar = 200 μm. ( B ) The expression of matrix metalloproteinases-2 (MMP2) and matrix metalloproteinases-9 (MMP9) were analyzed by Western blotting in the T24 cells. GAPDH was used as the loading control. ( C ) The concentration levels of MMP2 in the CM of T24 cells were analyzed by ELISA. ( D ) The protein activity of MMP9 in the CM of T24 cells were analyzed by the Fluorokine assay. * p < 0.05; ** p < 0.01 compared with the control group. # p < 0.05 compared to the FXR-O group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Incubation, Staining, Control, Expressing, Western Blot, Concentration Assay, Enzyme-linked Immunosorbent Assay, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of the overexpression of FXR on angiogenesis. ( A ) The human umbilical vein endothelial cells (HUVECs) were cultured with an FXR overexpression (72 h) conditioned medium (CM) and control CM of bladder cancer cells for 6 h. The formation of endothelial cell networks was observed and the number of branch points and tube length in the TSGH8301 and T24 CM were analyzed. The bar graphs show the quantitative results of relative branch points and tube lengths. Scale bar = 200 μm. ( B ) The concentration levels of vascular endothelial growth factor (VEGF) in the CM of TSGH8301 and T24 cells were analyzed by ELISA. ( C ) RT-PCR was used to analyze the mRNA expression of VEGF after FXR overexpression in TSGH8301 and T24 cells. * p < 0.05; ** p < 0.01; *** p < 0.001 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Cell Culture, Control, Concentration Assay, Enzyme-linked Immunosorbent Assay, Reverse Transcription Polymerase Chain Reaction, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of the overexpression of FXR on angiogenic related protein expression. ( A ) The expression of VEGFA, signal transducer and activator of transcription 3 (STAT3), phospho-Stat3 (p-STAT3), nitric oxide synthase 2 (NOS2) and hypoxia-inducible factors-1α (HIF-1α) were analyzed by Western blotting in TSGH 8301 and T24 cells after FXR overexpression. GAPDH was used as the loading control. ( B ) The bar graphs show the relative quantitative analysis of the aforementioned proteins. * p < 0.05; ** p < 0.01 compared with the control group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Expressing, Western Blot, Control

Journal: International Journal of Molecular Sciences

Article Title: Enhancement of Farnesoid X Receptor Inhibits Migration, Adhesion and Angiogenesis through Proteasome Degradation and VEGF Reduction in Bladder Cancers

doi: 10.3390/ijms23095259

Figure Lengend Snippet: The effect of proteosome inhibitor MG132 and lysosome inhibitor NH4Cl on the angiogenic abilities after FXR overexpression. ( A ) The human umbilical vein endothelial cells (HUVECs) were cultured with FXR overexpressed conditioned medium (CM) and control CM with or without MG132 or NH4Cl of bladder cancer cells for 6 h. The formation of an endothelial cell network was observed and the number of branch points and tube length in the T24 CM were analyzed. Scale bar = 200 μm. ( B ) VEGFA, p-STAT3, NOS2 and HIF-1α were analyzed by Western blotting in T24 cells with or without MG132 or NH4Cl. GAPDH was used as the loading control. The bar graphs show the relative attenuation of branch points and tube lengths. * p < 0.05, ** p < 0.01 compared with control group; # p < 0.05, ## p < 0.01; ### p < 0.001 compared with FXR overexpression group.

Article Snippet: RT4 and T24 cells were obtained from the Bioresource Collection and Research Center (BCRC), Taiwan, and grown in McCoy’s 5a medium.

Techniques: Over Expression, Cell Culture, Control, Western Blot

Journal: Scientific Reports

Article Title: Amyloids in bladder cancer hijack cancer-related proteins and are positive correlated to tumor stage

doi: 10.1038/s41598-025-88307-7

Figure Lengend Snippet: Determination of amyloid presence in bladder cancer cell lines by spectrofluorimetry ( a - c ) and confocal microscopy ( d - g ). ( a ) ThT fluorescence fold change (mean ± SD) was plotted versus ThT concentration (µM) for serial dilutions (1.6 to 0.1 mg/mL) of protein homogenates from RT4, EJ138 and HT1197 cell lines. The scale of the x-axis is expressed as Log2. ( b ) ThT fluorescence fold change (mean ± SD) was plotted against protein concentration (mg/mL) for ThT serial dilutions (200 to 3.125 µM) for RT4, EJ138 and HT1197 cell lines. ( c ) Spectrofluorometric analysis of ThT (50 µM final concentration) fluorescence fold change of cell homogenates (at 1.6 mg/mL) of each indicated cell type was measured. Letters denote statistical differences between groups. ( d ) RT4, EJ138 and HT1197 cells were stained with ThT (green) to visualize amyloids. F-actin (red) was stained with fluorescent phalloidin to visualize the cell border. Images are representative areas of maximum intensity Z-projections of serial sections at 0.25 μm. Arrows: intracellular/intercellular droplet-like ThT staining. Asterisk: fibril extracellular ThT staining. ( e ) Single focal plane of primary urothelial cells stained with ThT (green) and fluorescent phalloidin (red). ( f ) Single focal plane of blood cells from healthy donors stained with ThT (green), fluorescent phalloidin (red) and TO-PRO-3 iodide (white). g: granulocyte. e: erythrocyte. ( g ) Single focal plane of RT4, EJ138, HT1197 and primary urothelial cells stained with ThT (green), anti-oligomer antibody (red) and fluorescent phalloidin (white) to visualize the cell border. Images shown were taken at identical microscope settings. Bar: 10 μm.

Article Snippet: RT4 (ECACC 91091914), EJ138 (ECACC 85061108) and HT1197 (ECACC 87032403) cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECCAC).

Techniques: Confocal Microscopy, Fluorescence, Concentration Assay, Protein Concentration, Staining, Microscopy

Journal: Scientific Reports

Article Title: Amyloids in bladder cancer hijack cancer-related proteins and are positive correlated to tumor stage

doi: 10.1038/s41598-025-88307-7

Figure Lengend Snippet: Electrophoretic and size exclusion chromatographic analyses of cell line amyloids. ( a ) ThT staining of a 1.5% agarose gel native electrophoresis of protein homogenates treated with proteinase K (+ PK) or not treated (-PK). Protein homogenates from blood cells, RT4, EJ138, and HT1197 cell lines were included. The loaded sample in each well is indicated above each lane. Asterisks indicate the presence of positive ThT bands, which appear more evident in the signal on the weaker fluorescent background smear. ( b ) The same gel as in a , stained with Coomassie Blue. ( c ) Chromatogram obtained during gel filtration of HT1197 protein homogenate mixed with ThT, using Superdex matrix 200 (range: 3-600 kDa), recorded with in-line PDA (absorbance at 280 nm, 10 − 3 AU, blue line) and FD (Ex350 nm and Em472 nm, mV, black line). X-axis is elution volume (mL). Arrows indicate void volume (Vo) and total volume (Vt). Asterisks indicate ThT-positive peaks.

Article Snippet: RT4 (ECACC 91091914), EJ138 (ECACC 85061108) and HT1197 (ECACC 87032403) cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECCAC).

Techniques: Staining, Agarose Gel Electrophoresis, Electrophoresis, Filtration

Journal: Scientific Reports

Article Title: Amyloids in bladder cancer hijack cancer-related proteins and are positive correlated to tumor stage

doi: 10.1038/s41598-025-88307-7

Figure Lengend Snippet: Analysis of cell line amyloids purified by ultracentrifugation. ( a ) Microscopic analysis of purified amyloids from RT4, EJ138 and HT1197, stained with ThT and visualized by fluorescence microscopy, Congo red and visualized by polarization microscopy or uranyl acetate and visualized by transmitted electron microscopy. The arrows in each image show examples of the green-yellow colour of the amyloids when they are stained with Congo red and observed under polarized light. Blood samples from healthy donors subjected to the same ultracentrifugation protocol were included as a negative control for each staining. Bars: 1 μm. ( b ) Far-UV CD analysis of amyloids derived from EJ138 and HT1197. The black line represents the data obtained, while the red trace corresponds to the best fit from the analysis performed with the BeStSel web server ( https://bestsel.elte.hu/ ), which indicates that EJ138 and HT1197 have β-sheet contents of 34.2% and 35.5%, respectively. The inset shows the residuals of the fit. ( c ) Dynamic Light Scattering (DLS) analysis of amyloids derived from RT4, EJ138 and HT1197 cell lines. The left panel illustrates the highly reproducible autocorrelation functions obtained in triplicate for each batch, while the right panel depicts representative intensity-weighted size distributions. The hydrodynamic radius (RH) is plotted on the x-axis, and the Z-average (Zave) and polydispersity index (PdI), calculated as the mean of three replicates, are indicated alongside each distribution. ( d ) Differential Scanning Fluorimetry (DSF) obtained with amyloids from RT4, EJ138 and HT1197 cell lines. The ratio of intrinsic fluorescence measured at 350 and 330 nm upon heating and cooling scans is displayed in the left and right panels, respectively.

Article Snippet: RT4 (ECACC 91091914), EJ138 (ECACC 85061108) and HT1197 (ECACC 87032403) cell lines were obtained from the European Collection of Authenticated Cell Cultures (ECCAC).

Techniques: Purification, Staining, Fluorescence, Microscopy, Electron Microscopy, Negative Control, Derivative Assay