urothelial cell line 5637  (ATCC)

Journal: iScience

Article Title: IFRD1 is required for maintenance of bladder epithelial homeostasis

doi: 10.1016/j.isci.2024.111282

Figure Lengend Snippet: Loss of IFRD1 causes alterations in urothelial cellular architecture and molecular pathways (A) Heatmap of the global tissue transcriptome of WT and Ifrd1 −/− bladders with fold change >1.5 and Benjamini-Hochberg FDR-adjusted p < 0.05 ( n = 4 bladders/group). (B) GSEA Hallmark pathway analysis showing significantly upregulated pathways in the WT versus Ifrd1 −/− (blue bars) and Ifrd1 −/− versus WT (red bars) bladders below FDR threshold of 0.25. (C) H&E staining shows vesicles in WT (highlighted in black circles) and accumulated vesicles in Ifrd1 −/− bladders in the superficial cells. Scale bar, 50 μm. (D) Transmission electron microscopy (TEM) of superficial cells show MVB (red arrows) in WT and Ifrd1 −/− . Ifrd1 −/− show accumulation of endolysosomes (white arrows). Magnified insets show the endolysosomes. Blue circles highlight the endolysosomes. Scale bar, 2 μm. (E) Analysis of number of endolysosomes per cross-section per 100 μm 2 in WT and Ifrd1 −/− superficial cells ( n = 40–97 TEM images/group from 2 WT mice and 3 Ifrd1 −/− mice; data shown as mean (±SEM); p value by Mann-Whitney test). (F) Immunostaining of UPK3A (green) in WT and Ifrd1 −/− superficial cell layer ( n = 6 bladders/group). Magnified insets highlight the apical staining pattern in superficial cell in the WT versus diffused staining pattern in the Ifrd1 −/− . Scale bar, 50 μm. (G) Real-time quantitative PCR (RT-qPCR) analysis of Upk3a transcripts in WT and Ifrd1 −/− bladders ( n = 9 bladders/group; data shown as mean (±SD); p value by Mann-Whitney test).

Article Snippet: Human urinary urothelial carcinoma cell line (5637, HTB9) (ATCC) was cultured in RPMI 1640 media with 10% fetal bovine serum in a humidified atmosphere at 37°C with 5% CO2.

Techniques: Staining, Transmission Assay, Electron Microscopy, MANN-WHITNEY, Immunostaining, Real-time Polymerase Chain Reaction, Quantitative RT-PCR

Journal: iScience

Article Title: IFRD1 is required for maintenance of bladder epithelial homeostasis

doi: 10.1016/j.isci.2024.111282

Figure Lengend Snippet: Ifrd1 −/− mice display increased urothelial cell death, dysregulated renewal and aberrant voiding behavior (A) TUNEL staining (green) of WT and Ifrd1 −/− urothelial cells. Nuclei are stained with DAPI (blue) ( n = 3/group). Scale bar, 100 μm. (B) DHE staining (red) of WT and Ifrd1 −/− urothelium. Nuclei are stained with DAPI (blue). Quantitation of DHE staining intensity ( n = 3 bladders/group; data shown as mean (±SD); p values by unpaired t test). Scale bar, 100 μm. (C) Representative TEM (left) and urine cytology (right) images show exfoliated bladder epithelial cells. Quantitation of exfoliated/shed epithelial cells ( n = 5 WT, 4 Ifrd1 −/− mice; data shown as mean (±SD); p values by unpaired t test). Scale bar, 2 μm. (D) Immunostaining for Ki-67 (red) and E-cadherin (green) in WT and Ifrd1 −/− urothelial cells ( n = 3/group). Scale bar, 100 μm. (E) Immunostaining of CDKN1B (red) in WT and Ifrd1 −/− urothelium ( n = 3/group). Scale bar, 50 μm. (F) RT-qPCR analysis of Cdkn1b transcripts in WT and Ifrd1 −/− bladders ( n = 6 WT, 3 Ifrd1 −/− bladders; data shown as mean (±SD); p values by Mann-Whitney test). (G) FITC-dextran permeability assay (green) in the WT and Ifrd1 −/− urothelial cells reveal superficial cells with intracellular FITC-dextran in the Ifrd1 −/− urothelium. Nuclei are stained with DAPI (blue) ( n = 3/group). Quantitation of FITC-dextran staining intensity ( n = 3 bladders/group; data shown as mean (±SD); p values by Mann-Whitney test). Scale bar, 100 μm. (H) Representative images of WT (left) and Ifrd1 −/− (right) void spot assay (VSA). (I) Quantitation of the area of total voids from the VSA. Each dark dot is the average area of all the individual voids that are depicted as transparent dots of the same color. ( n = 4/group, data shown as mean (±SEM), p values by unpaired t test). (J) Quantitation of the size of all the individual spots from the VSA. All the data points from one mouse are presented in one color. (Data represented as interquartile range of size distribution where the central line represents the median (±SD), n = 4/group, p values by unpaired t test).

Article Snippet: Human urinary urothelial carcinoma cell line (5637, HTB9) (ATCC) was cultured in RPMI 1640 media with 10% fetal bovine serum in a humidified atmosphere at 37°C with 5% CO2.

Techniques: TUNEL Assay, Staining, Quantitation Assay, Immunostaining, Quantitative RT-PCR, MANN-WHITNEY, FITC-Dextran Permeability Assay, Spot Test