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