primary bladder urothelial cells  (ATCC)

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Differentially expressed genes (DEGs) in full-thickness bladder tissues from HIC patients and controls identified via RNA sequencing (|log₂FC|≥2; adjusted p < 0.05, two-sided Wald test using DESeq2). Gene Ontology (GO) ( B ) and KEGG ( C ) enrichment analyses of DEGs (two-sided hypergeometric test with FDR correction). D UMAP visualization of single-cell transcriptomic landscape of urothelium, with urothelial cells (UCs) identified by KRT19 and UPK1A expression. GO ( E ) and KEGG ( F ) analyses of upregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). GO ( G ) and KEGG ( H ) analyses of downregulated DEGs in UCs (two-sided hypergeometric test with FDR correction). I , J Gene Set Enrichment Analysis (GSEA) of altered pathways in UCs (two-sided permutation test with 1000 permutations, with multiple testing corrected using the FDR). The exact p value for TLR signaling of UCs in panel I is 0.0009. K Expression of Toll-like receptor (TLR) subtypes in UCs from HIC bladders. mRNA ( L ) and protein ( M ) expression levels of TLR3 in isolated urothelium from control and HIC patients ( n = 7 control vs. 10 HIC; data show median (IQR); two-sided Mann–Whitney U test; ns not significant; Bar for panel M: 1 cm). N Immunostaining showing the distribution of TLR3 protein in the urothelium of patients with HIC ( n = 7 control vs. 10 HIC; one section and field per patient; Bar: 100 μm). IHC immunohistochemistry, HL Hunner lesions, U urothelium, LP lamina propria (blue line), M muscularis. Source data are provided as a Source Data file.

Article Snippet: Primary bladder urothelial cells (ATCC, Cat. PCS-420-010) were cultured in bladder epithelial cell basal medium (ATCC, Cat. PCS-420-032) supplemented with bladder epithelial growth kit (ATCC, Cat. PCS-420-042) at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: RNA Sequencing, Expressing, Isolation, Control, MANN-WHITNEY, Immunostaining, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Fecal microbiota transplantation (FMT) in antibiotic-pretreated mice (Abx-mice), using stool samples from healthy controls (Group A, No. of controls = 5) and HIC patients (Group B, No. of patients = 5). B Bladder weight comparison post-FMT. C Assessment of voiding behavior in FMT-recipient mice (each mouse measured once). D Evaluation of mechanical pain threshold following FMT. Comparisons: (1) Control vs. Autoimmune cystitis (AC); (2) Control vs. Control + A; (3) Control vs. Control + B; (4) AC vs. AC + A; (5) AC vs. AC + B. E Representative histological analysis of bladder tissues after FMT (one section and field per mouse). F Quantification of urinary TCDCA and TUDCA in recipient mice. For panels ( B – F ): group sizes are n = 7 for Control + A and n = 8 for all other groups; data are presented as median (IQR); statistical analysis was performed using the Kruskal–Wallis test; ns not significant. IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: Primary bladder urothelial cells (ATCC, Cat. PCS-420-010) were cultured in bladder epithelial cell basal medium (ATCC, Cat. PCS-420-032) supplemented with bladder epithelial growth kit (ATCC, Cat. PCS-420-042) at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Transplantation Assay, Comparison, Control, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A Experimental workflow of E. avium colonization in antibiotic-pretreated mice (Abx-mice, n = 6 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . B Increased abundance of E. avium confirmed by metagenomic sequencing ( n = 6 per group). C Evaluation of voiding behavior following E. avium transplantation ( n = 6 per group; one measurement per mouse). D Measurement of mechanical pain threshold post-transplantation ( n = 6 per group). E Representative histological analysis of bladder tissues after E. avium transplantation ( n = 6 per group; one section and field per mouse). F , G Quantification of blood and urinary bile acids using comprehensive targeted bile acid profiling ( n = 6 per group). H Cell viability of human urothelial cells (HUCs) following TCDCA or TUDCA treatment ( n = 3 independent experiments). I Expression of ZO-1, TNF-α, and TLR3 following TCDCA (200 µM) exposure ( n = 3 independent experiments). J Experimental workflow of intravesical instillation of TCDCA in rats ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . K Evaluation of voiding function after TCDCA instillation ( n = 5 per group; each rate measured once). L Assessment of mechanical pain threshold following TCDCA exposure ( n = 5 per group). M Representative histology of bladder tissues post-instillation ( n = 5 per group; one section per rat, one field quantified per section). For panels ( B – H , K , L ): data are presented as median (IQR). Statistical analysis was performed using the two-sided Mann–Whitney U test. IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: Primary bladder urothelial cells (ATCC, Cat. PCS-420-010) were cultured in bladder epithelial cell basal medium (ATCC, Cat. PCS-420-032) supplemented with bladder epithelial growth kit (ATCC, Cat. PCS-420-042) at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Sequencing, Transplantation Assay, Expressing, MANN-WHITNEY, Immunohistochemistry

Journal: Nature Communications

Article Title: Multi-omics analysis identifies a microbiota–bile acid–TLR signaling axis driving bladder injury in interstitial cystitis

doi: 10.1038/s41467-025-68060-1

Figure Lengend Snippet: A , B Expression of tight junction protein ZO-1 and inflammatory marker TNF-α after TLR3 intervention in TCDCA-pretreated human urothelial cells (HUCs) ( n = 3 independent experiments). C Transepithelial resistance (TER) changes following TLR3 intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). D , E Expression of ZO-1 and TNF-α after pentosan polysulfate sodium (PPS) administration in TCDCA-pretreated HUCs ( n = 3 independent experiments). F TER changes following PPS intervention in TCDCA-pretreated HUCs ( n = 3 independent experiments). G Experimental workflow showing TLR3 inhibitor and PPS administration to rats following intravesical TCDCA instillation ( n = 5 per group). Created in BioRender. Chen, J. (2025) https://BioRender.com/3lccwo1 . H Assessment of voiding function (one measurement per rat) and pain threshold after TLR3 inhibition ( n = 5 per group). I Representative histological images of bladder tissues post-TLR3 inhibition ( n = 5 per group; one section and field per rat). For panels ( B , C , E , F , H ): data are presented as median (IQR). Statistical analysis was performed using the Kruskal–Wallis test. ns not significant, IHC immunohistochemistry, Red arrow: urothelial thinning, detachment, and exposure, Blue line: LP lamina propria, U urothelium, M muscularis. Source data are provided as a Source Data file.

Article Snippet: Primary bladder urothelial cells (ATCC, Cat. PCS-420-010) were cultured in bladder epithelial cell basal medium (ATCC, Cat. PCS-420-032) supplemented with bladder epithelial growth kit (ATCC, Cat. PCS-420-042) at 37 °C in a humidified atmosphere of 95% air and 5% CO 2 .

Techniques: Expressing, Marker, Inhibition, Immunohistochemistry

Journal: bioRxiv

Article Title: Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

doi: 10.1101/2025.10.22.683857

Figure Lengend Snippet: (A) Mouse bladder organoids treated with OM-89 under different regimes (pre-application: OM-89 exposure for 72h and 24h rest period; co-application: OM-89 exposure together with antibiotic treatment; continuous application: OM-89 exposure both 72h before microinjection and throughout the entire experiment). After microinjection of fluorescently labelled UPEC (0h post-infection, pi), bacterial growth (4h pi), antibiotic-mediated killing (7h pi), and regrowth 3 hours after antibiotic withdrawal (10h pi) were monitored. (B)-(D) CFT073 signal 10h pi (3h regrowth) at 10x MIC in (B) pre-application, (C) co-application, and (D) continuous application regimes in mouse bladder organoids. Each dot represents one organoid. Values normalized to PBS control (dashed line). Mean ± 95% CI. Welch’s t test. N ≥ 15 per condition for (B), n ≥ 17 per condition for (C) and n ≥ 23 per condition for (D). Images show representative organoids (grey, surface reconstruction) and UPEC (magenta, some marked with arrows). (E) CFT073 signal 15h pi (8h regrowth) at 10x MIC in the continuous application regime in mouse bladder organoids. Values normalized to PBS control (dashed line). Mean ± 95% CI. Mann-Whitney test. N ≥ 15 per condition. Images show representative organoids (grey, surface reconstruction) and UPEC (magenta, some marked with arrows). (F)-(H) CFT073 signal 7h pi (killing) at 10x MIC in (F) pre-application, (G) co-application, and (H) continuous application regimes in mouse bladder organoids. Values normalized to PBS control (dashed line). Mean ± 95% CI. Welch’s t test for (F), Mann-Whitney test for (G), (H). N ≥ 15 per condition for (F), n ≥ 17 per condition for (G) and n ≥ 14 per condition for (H). (I) FITC-labelled ampicillin uptake into monolayers of mouse bladder epithelial cells during the continuous application regime. Quantification of fluorescence after background subtraction of PBS- or OM-89-treated cells without labelled antibiotic. Each dot represents one field of view. Mean ± 95% CI. Mann-Whitney test. N ≥ 7 per condition. Z-projection (maximum intensity) of representative images. FITC-ampicillin shown in cyan. (J) CFT073 signal 7h pi (1x MIC; killing and 10h pi (regrowth) in mouse bladder organoids during the continuous application regime. Each dot represents one organoid. Values normalized to PBS control (dashed line). Mean ± 95% CI. Mann-Whitney test. N ≥ 15 per condition. Images show representative organoids (grey, surface reconstruction) and UPEC (magenta, some marked with arrows).

Article Snippet: Mouse primary bladder epithelial cells were obtained from C57BL/6 mice (Charles River Laboratories) aged 3-4 months or female ROSA MT/MG mice (Jackson Laboratories) at age 4 months.

Techniques: Microinjection, Infection, Control, MANN-WHITNEY, Fluorescence

Journal: bioRxiv

Article Title: Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

doi: 10.1101/2025.10.22.683857

Figure Lengend Snippet: (A-C) Mouse bladder organoids treated with OM-89 under (A) pre-application, (B) co-application, or (C) continuous application regimes. Quantification of CFT073 fluorescence area at 4h post-infection (pi). Each dot represents one organoid. Values normalized to PBS control (dashed line). Mean ± 95% CI. Welch’s t test. N ≥ 15 per condition for (A), n ≥ 17 per condition for (B) and n ≥ 23 per condition for (C). (D) CFT073 signal over time in the continuous application regime. Quantification of bacterial fluorescence area normalized to PBS-treated organoids at the same timepoint. Each dot represents one organoid, lines connect matched organoids. RM two-way ANOVA with Šídák’s correction; p-value shown for treatment effect. N ≥ 14 per condition. (E), (F) Uptake of (E) FITC-labeled gentamicin and (F) Dextran-TMR in mouse bladder epithelial cell monolayers. Quantification of fluorescence after background subtraction of PBS- or OM-89-treated cells without labeled antibiotic. Mean ± 95% CI. Welch’s test. N = 8 per condition. Z-projection (maximum intensity) of representative images. FITC-gentamicin and Dextran-TMR shown in cyan

Article Snippet: Mouse primary bladder epithelial cells were obtained from C57BL/6 mice (Charles River Laboratories) aged 3-4 months or female ROSA MT/MG mice (Jackson Laboratories) at age 4 months.

Techniques: Fluorescence, Infection, Control, Labeling

Journal: bioRxiv

Article Title: Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

doi: 10.1101/2025.10.22.683857

Figure Lengend Snippet: Staining of differentiated mouse monolayer for bladder epithelial cell markers: Cytokeratin (CK) 20 – umbrella-like cells. Uroplakin (UP) 3a – umbrella-like cells. CK13 – intermediate cells. CK13-positive, binucleated cells indicate late intermediate cells. CK5 – basal cells. Binucleated cells indicated with asterisk.

Article Snippet: Mouse primary bladder epithelial cells were obtained from C57BL/6 mice (Charles River Laboratories) aged 3-4 months or female ROSA MT/MG mice (Jackson Laboratories) at age 4 months.

Techniques: Staining

Journal: bioRxiv

Article Title: Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

doi: 10.1101/2025.10.22.683857

Figure Lengend Snippet: (A)-(D), (F), (G) Quantification of vesicle numbers and size for (A) Rab11A, (B) Rab27B, (C) p62, (D) LC3b, (F) Rab7, and (G) Lamp1 in infected monolayers of mouse bladder epithelial cells with and without OM-89 treatment. Values normalized to PBS control (dashed line). Mean ± 95% CI. Mann-Whitney test for Rab11A and Rab7 (A), (F); Welch’s t test for Rab27B, p62, LC3b and Lamp1 (B)-(D), (G). N = 12 per condition. Z-projection (maximum intensity) of representative images. Foci, cyan; DAPI, grey. (E) Western blot analysis of LC3b during infection of monolayers. LC3b-II/Gapdh ratio normalized to PBS control (dashed line). Mean ± 95% CI. Kruskal-Wallis test with Dunn’s correction. Autophagic flux (BafA1-blocked minus unblocked LC3b-II/Gapdh): PBS = 24.75 ± 10.68; OM-89 = 65.87 ± 9.11. N = 3 per condition.

Article Snippet: Mouse primary bladder epithelial cells were obtained from C57BL/6 mice (Charles River Laboratories) aged 3-4 months or female ROSA MT/MG mice (Jackson Laboratories) at age 4 months.

Techniques: Infection, Control, MANN-WHITNEY, Western Blot

Journal: bioRxiv

Article Title: Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

doi: 10.1101/2025.10.22.683857

Figure Lengend Snippet: (A) GeneBridge MMAS (Module-Module Association Score) analysis of KEGG lysosomal terms in human bladder datasets. Positively correlated modules linked to immune responses and bacterial defense are highlighted. (B) Quantification of vesicle numbers and size of LysoTracker DND-99 in infected monolayers of mouse bladder epithelial cells with and without OM-89 treatment. Values normalized to PBS controls (dashed line). Mean ± 95% CI. Vesicle numbers analyzed with Welch’s t test; vesicle size with Mann-Whitney test. N = 12 per condition. (C) Intraorganelle pH in infected monolayers with and without OM-89 treatment. Mean ± 95% CI. Welch’s t test. N = 12 per condition. (D)-(G) Co-localization of intracellular UPEC with (D) LysoTracker, (E) Lamp1, (F) Cathepsin L and (G) Cathepsin L with bafilomycin A1 (BafA1) blocking in monolayers of mouse bladder epithelial cells. Intensities normalized to ROI area. Mann-Whitney test. N ≥ 24 per condition for LysoTracker, n ≥ 18 per condition for Lamp1, n ≥ 19 per condition for Cathepsin L, n ≥ 15 per condition for Cathepsin L + BafA1 blocking. Enrichment score (= observed overlap/random overlap) between OM-89 vs PBS for (D) 0.68 vs 0.71, (E) 0.94 vs 0.89, (F) 1.25 vs 0.91 and (G) 1.06 vs 1.17. Random overlap from , F. Z-projection (maximum intensity) of representative images. (D), (E) Phalloidin, yellow; (F), (G) CellMask, yellow; (D), (E) DAPI, grey; (F), (G) Hoechst, grey; (D)-(G) UPEC, magenta; LysoTracker/Lamp1/Cathepsin L, cyan. Insets: intracellular UPEC (highlighted squares) with ROIs used for intensity quantification (scale bar of inserts, 2 µm). (H), (I) CFT073 signal 7h pi (killing) and 10 h pi (regrowth) in mouse bladder organoids after blocking with (H) BafA1 or (I) CQ. Each dot represents one organoid. Values normalized to PBS control (dashed line). Mean ± 95% CI. Mann-Whitney test. N = 15 per condition. Images show representative organoids (grey, surface reconstruction) and UPEC (magenta, some marked with arrows).

Article Snippet: Mouse primary bladder epithelial cells were obtained from C57BL/6 mice (Charles River Laboratories) aged 3-4 months or female ROSA MT/MG mice (Jackson Laboratories) at age 4 months.

Techniques: Infection, MANN-WHITNEY, Blocking Assay, Control

Journal: bioRxiv

Article Title: Epithelial Reprogramming by OM-89 Enhances Antibiotic Clearance of Uropathogenic E. coli in a Bladder Organoid Model

doi: 10.1101/2025.10.22.683857

Figure Lengend Snippet: (A) Intraorganelle pH in uninfected monolayers with and without OM-89 treatment. Mean ± 95% CI. Mann-Whitney test. N = 12 per condition. (B)-(D), (F) Co-localization of random regions of interest (ROIs) with (B) LysoTracker, (C) Lamp1, (D) Cathepsin L and (F) Cathepsin L with bafilomycin A1 (BafA1) in monolayers of mouse bladder epithelial cells. Intensities normalized to ROI area. (B), (D), (F) Mann-Whitney test. (C) Welch’s test. N ≥ 24 per condition for (B), n ≥ 18 per condition for (C), n ≥ 19 per condition for (D) and n ≥ 16 per condition for (F). Z-projection (maximum intensity) of representative images. (B), (C) Phalloidin, yellow; (D), (F) CellMask, yellow; (B), (C) DAPI, grey; (D), (F) Hoechst, grey; (B)-(D), (F) UPEC, magenta; LysoTracker/Lamp1/Cathepsin L, cyan. Insets: random ROIs used for intensity quantification (scale bar of inserts, 2 µm). Data correspond to . (E) Cathepsin L activity during infection with or without OM-89, and with BafA1 or chloroquine (CQ) blocking. Values normalized to unblocked PBS controls (dashed line). Mean ± 95% CI. Brown-Forsythe ANOVA with Welch’s correction. N = 10 per condition. (G), (H) CFT073 growth after (G) BafA1 and (H) CQ blocking. Quantification of bacterial fluorescence area inside organoids 4h pi. Each dot represents one organoid. Values normalized to PBS control (dashed line). Mean ± 95% CI. Mann-Whitney test. N ≥ 15 per condition for (G) and n ≥ 19 per condition for (H).

Article Snippet: Mouse primary bladder epithelial cells were obtained from C57BL/6 mice (Charles River Laboratories) aged 3-4 months or female ROSA MT/MG mice (Jackson Laboratories) at age 4 months.

Techniques: MANN-WHITNEY, Activity Assay, Infection, Blocking Assay, Fluorescence, Control