human bladder cancer cell lines ht1197  (ATCC)

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: (a) Overview of medicinal chemistry strategy for covalent PPARG inverse agonist optimization resulting in FTX-6746. (b) Crystal structure of FTX-6746 covalent adduct with PPARG LBD. The PPARG LBD (gray) adopts a similar conformation to that observed with T0070907 (PDB 6ONI) with H12 (orange) inserted into the ligand binding pocket with the C-terminal carboxylate forming a key H-bond with the quinolone nitrogen of the FTX-6746 adduct (yellow dashes). (c) Dose-dependent recruitment of NCOR1 ID2 to RXRA S427F :PPARG heterodimers, demonstrating the superiority of FTX-6746 for countering the activation bias driven by this mutation over T0070907 and SR10221. Representative curves from ≥ 3 biological replicates are shown. Data points indicate the average of technical duplicates from a representative biological replicate. The 95% confidence interval for EC 50 values of SR10221, T0070907 and FTX-6746, respectively, were determined to be: 109 nM ± 30 nM, 60.5 ± nM, 197.5 ± 8.5 nM. The 95% confidence interval for the E max values relative to T0070907 for SR10221 and FTX- 6746, respectively, were determined to be: 6.2 ± 0.6% and 170.5 ± 2%. Rosiglitazone was not present for these experiments. (d) Comparison of SR10221, T007097 and FTX-6746 ability to suppress PPARG target gene ANGPTL4 in RXRA WT (blue, 5637 cell line) and RXRA S427F (orange, HT1197 cell line) contexts. Data indicate the activation bias observed biochemically reduces T0070907 and SR10221 efficacy and the superior repressive biasing of FTX-6746 enables it to achieve similar repressive effects in both contexts. Data is representative of two separate biological experiments.

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Ligand Binding Assay, Activation Assay, Mutagenesis, Comparison

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: (A) Selective engagement of FX-909 across urothelial carcinoma cell lines. Global cysteine profiling and engagement of FX-909 in UMUC9, HT1197 and 5637 cell lines following 30 min treatment. PPARG C313 was the only significantly engaged target (orange). Competition ratio (CR) was calculated dividing the control channel (DMSO) by the electrophile treated channel. The blue dotted line indicates a CR of 3. (B) Cellular potency of FX-909. (top) Dose-dependent suppression of IL1B expression by FX-909 in the urothelial carcinoma line UMUC9. Relative IL1B expression and IC 50 value are indicated. (bottom) Dose-dependent suppression of ANGPTL4 expression by FX-909 in the urothelial carcinoma line HT1197. Relative ANGPTL4 expression and IC 50 value are indicated. (C) Transcriptional changes induced by PPARG inverse agonists in UMUC9, HT1197 and 5637 cell lines following 24 hr treatment. (top) Heatmap showing the effect of T0070907, FTX-6746 and FX-909 on the genes that are differentially up- or down-regulated by FX-909 (see ). (bottom) Comparison of gene expression changes induced by FX-909 vs T0070907. Each dot corresponds to a gene that showed statistically significant change in response to T0070907, FTX- 6746 or FX-909 (n=6,759 genes; FDR < 0.05) as assessed from a mixed effect model where cell lines are treated as a random effect. Red line corresponds to a linear regression model with a slope of β=1.8, indicating that on average FX-909 induces 3.5=fold (2^1.8) times greater fold changes in gene expression. Dashed black line shows where data would fall if FX-909 and T0070907 induced effects of the same magnitude. The lack of gene expression changes in the bottom right and top left quadrants of the plot indicates that FX-909 does not regulate different genes from T0070907.

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Control, Expressing, Comparison, Gene Expression

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: Selective engagement of FTX-6746 across urothelial carcinoma cell lines. Global cysteine profiling and engagement of FTX-6746 in UMUC9, HT1197 and 5637 cell lines following 30 min treatment. PPARG C313 was the only significantly engaged target (green). Competition ratio (CR) was calculated dividing the control channel (DMSO) by the electrophile treated channel. The blue dotted line indicates a CR of 3.

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Control

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: (A) Principal component analysis (PCA) of genome-wide expression changes (on log2 scale) induced by PPARG inverse agonists in UMUC9, HT1197 and 5637 cell lines after 24 hr treatment. (B) Consensus FX-909 induced differential gene expression was assessed using a mixed effect model where Treatment was a fixed effect and Cell Line was a random effect. Differentially expressed genes were determined by contrasting the FX-909 treatment with DMSO and using the cutoffs of FDR < 0.05 and absolute log2(fold change) > 0.6.

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Genome Wide, Expressing, Gene Expression

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: (A) Phenotypic potency of PPARG inverse agonists. GI 50 values of UC cell lines treated with T0070907, FTX-6746 or FX-909 for 11-14 days. GI 50 values were calculated relative to the DMSO treated cells using a crystal violet stain quantitation. GI 50 values greater than the maximum dose tested - 500 nM (FX-909) or 1500 nM (T0070907 and FTX-6746) - were graphed as 500 nM and 1500 nM, respectively. * = PPARG/RXRA mutant or PPARG amplified lines. Data are a representative of multiple individual experiments performed. (B) Phenotypic potency of FX-909 in WT or C313A isogenic HT1197 cell lines. Treatment of parental and C313A HT1997 cells lines for 14 days with FX-909 resulted in potent growth inhibition only in parental line, growth curve on left and images of clonogenic assay on right.

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Staining, Quantitation Assay, Mutagenesis, Amplification, Inhibition, Clonogenic Assay

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: Clonogenic growth assay of HT1197 cells treated with FTX-6746, FX-909 or T0070907. Percent cell density relative to DMSO control well shown, data are representative of three independent experiments.

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Growth Assay, Control

Journal: bioRxiv

Article Title: Discovery and characterization of FX-909, a covalent inverse agonist of PPARG rationally designed to impose a powerful repressive bias in PPARG for the treatment of PPARG/RXRA-activated muscle-invasive urothelial cancers

doi: 10.1101/2025.05.08.652848

Figure Lengend Snippet: – (A) UMUC9 xenograft efficacy study shows FX-909 elicits durable tumor regression at all doses, while FTX-6746 elicits tumor regression that is not durable upon drug cessation. (B) HT1197 xenograft efficacy study shows tumor regression for both FX-909 and FTX-6746. (C) PK/PD relationship for FTX-6746 and FX-909. (D) H&E staining of adipose tissue from mice in study shown in (A).

Article Snippet: The UC cell lines HT1197 (CRL1473), 5637 (HTB-9), HT1376 (CRL-1472), J82 (HTB- 1), RT4 (HTB-2) and SW780 (CRL-2169) were purchased from ATCC.

Techniques: Staining

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

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 proteome of RT4 and HT1197 cell lines. ( a ) Aggregation-prone analysis of the first MS-identified proteins for each cell line. Identifiers, name, and molecular weights of identified proteins are indicated, as well as the number of identified peptides and the sequence coverage of each protein. MS-identified-peptides containing amyloid-prone regions are indicated in bold letters, also related to no amyloid-prone identified peptides by percentages. ( b , c ) KEGG Mapper analysis of amyloid proteins in RT4 ( b ) and HT1197 ( c ). Pathways are depicted by decreasing number of genes in each one. ( d ) Venn diagram showing the overlap between amyloid-identified proteins of RT4 and HT1197 cell lines and tumor suppressor (TumSupp) and/or oncogenes (Onc). Numbers in parentheses indicate the total number of elements in each set. Protein identity for elements in selected overlaps is indicated. ( e ) Venn diagram showing the overlap between amyloid-identified proteins of RT4 and HT1197 cell lines and bladder cancer driver genes (BCDriver). Numbers in parentheses indicate the total number of elements in each set. ( f ) Dot blot analysis with anti-HDAC1 and anti-RhoA antibodies on purified amyloids derived from RT4 and HT1197 cells. Prior to being spotted on the membrane, the purified amyloids were dissolved in either water or formic acid, as indicated. Blood was included as a control. The images of the blots were subjected to cropping in order to enhance clarity. The blots in their original state are presented in Supplementary Fig. 3.

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: Sequencing, Dot Blot, Purification, Derivative Assay, Membrane, Control

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: PGK1 ( a ), HDAC1 ( b ) and RhoA ( c ) distribution in RT4 and HT1197 cells. Images are single focal planes at intracellular level of immunocytochemistry experiments showing the distribution of each protein antibody signal (red) on RT4 and HT1197 cells treated or not treated with 70% formic acid. ThT (green) and fluorescent phalloidin (white) were included as counterstain probes. Arrows: extracellular fibrillar signal; arrowhead: extracellular fibrillary signal in FA treated cells; asterisk: intracellular signal in FA treated cells. 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: Immunocytochemistry

Journal: iScience

Article Title: Multilevel plasticity and altered glycosylation drive aggressiveness in hypoxic and glucose-deprived bladder cancer cells

doi: 10.1016/j.isci.2025.111758

Figure Lengend Snippet: BLCA cells exhibit remarkable tolerance to hypoxia and low glucose, adopting a quasi-quiescent and more aggressive invasive behavior (A) Hypoxia or low glucose (normoxia-Glc) significantly upregulates HIF-1α expression in BLCA cell lines, which is further enhanced when combined (hypoxia-Glc). (B) BLCA cells cultured in hypoxia and low glucose produce residual levels of lactate. Individually, these stressors induce the production of lactate. (C) Hypoxia and low glucose significantly suppress cell proliferation. Individually, low oxygen or low glucose inhibits cell proliferation. The combination of these stressors further exacerbates this effect in all cell lines. (D) BLCA cells maintain their viability under hypoxia and low glucose. The combined environmental stress from hypoxia and low glucose does not significantly impact the viability of 5637 and T24 cells. RT4 and HT1197 cells exhibit a 30%–45% reduction in viability under these conditions, suggesting a limited adaptive capacity. (E) BLCA cells display increased invasiveness under hypoxia or low glucose. This is significantly potentiated when both stimuli are combined. (F) BLCA cells demonstrate remarkable adaptability to microenvironmental changes with minimal impact on cell viability. Restoring oxygen and glucose levels does not affect cell viability, underscoring the high plasticity of these cells to endure drastic microenvironmental changes. (G) BLCA cells restore basal proliferation after 48 h of reoxygenation with glucose restoration. Both 5637 and T24 cells regain proliferative capacity, fully reinstating proliferation after 48 h, highlighting their plasticity in responding to microenvironmental challenges. (H) After 24 h of reoxygenation with glucose restoration, BLCA cells exhibit a significant reduction in invasion, which is fully restored under normoxia after 48 h. (I) Hypoxia and low glucose increase T24 cells' resistance to cisplatin across a wide range of concentrations, including its IC50, whereas 5637 cells remain unchanged. Error bars represent mean ± SD for three independent experiments. One-way ANOVA followed by Tukey’s multiple comparison test and the Mann-Whitney test were used for statistical analysis. Results were considered statistically significant when p < 0.05.

Article Snippet: Human BLCA cell line HT1197 (white male patient) , ATCC , CRL-1473 TM.

Techniques: Expressing, Cell Culture, Comparison, MANN-WHITNEY

Journal: iScience

Article Title: Multilevel plasticity and altered glycosylation drive aggressiveness in hypoxic and glucose-deprived bladder cancer cells

doi: 10.1016/j.isci.2025.111758

Figure Lengend Snippet: BLCA cell lines under hypoxia and low glucose experience profound transcriptome remodeling, linked to the acquisition of more aggressive phenotypes, which is supported by the poor prognosis observed in TCGA-BLCA patients (A) BLCA cell lines under hypoxia and low glucose display distinct transcriptomes but share common responses to these conditions. PCA for transcriptomics data reveals that PC1 (94% variance) primarily distinguishes differences between cell lines, whereas PC2 (5% variance) highlights marked changes between normoxic and stressed cells. (B) The volcano plot showcases global transcriptional changes between normoxia and hypoxia plus low glucose. Exposure to these stressors alters the expression of 4,044 genes (1,722 upregulated, 2,322 downregulated), indicating significant transcriptome remodeling. (C) Bi-clustering heatmap of the top 30 differentially expressed genes illustrates co-regulation under stress, supporting proliferation arrest, resistance to cell death, and invasion. Heatmap plots log2 transformed expression values of genes in samples. (D) Enrichment analysis of GO terms for differentially expressed genes reveals alterations in key pathways associated with cell-cell adhesion, cell proliferation, and resistance to cell death. (E) Prognostic evaluation identifies a hypoxia and glucose-deprivation-linked four-gene signature ( TAGLN high ; SLC2A3 high ; TRIB3 high ; TMEM158 high ). Univariate Cox regression analysis of the top 30 differentially expressed genes identifies seven genes associated with OS. Higher expression levels of four genes, upregulated under hypoxia and low glucose, significantly correlate with poor OS, constituting a stress signature. (F) Validation of the prognosis significance of the hypoxia-related four-gene signature in BLCA patients from TCGA. Kaplan-Meier curves of OS and PFS show significantly worse clinical outcomes for patients displaying the stress-related gene signature compared to the remaining patients in the cohort. (G) Bi-clustering heatmap showing the association between the stress-related signature and bladder tumors. Heatmap plots log2 transformed expression values of the four hypoxia-related differentially expressed genes, showing clear differentiation between cancer and healthy bladder samples.

Article Snippet: Human BLCA cell line HT1197 (white male patient) , ATCC , CRL-1473 TM.

Techniques: Expressing, Transformation Assay, Biomarker Discovery

Journal: iScience

Article Title: Multilevel plasticity and altered glycosylation drive aggressiveness in hypoxic and glucose-deprived bladder cancer cells

doi: 10.1016/j.isci.2025.111758

Figure Lengend Snippet: Hypoxia and low glucose shift BLCA cell metabolism from glycolytic to lipolytic, increasing lipid droplet formation and reducing the number of active mitochondria (A–C) PLS-DA analysis reveals similar metabolic responses in 5637 and T24 cells under microenvironmental stress (A). Volcano plot highlights significant metabolome alterations in response to hypoxia and low glucose (B). Downregulated metabolites include UDP-Glc, UDP-GalNAc, gluconic acid, and citric acid, whereas increased metabolites indicate active fatty acid transport and β-oxidation (C). Significant reduction in key metabolites linked to nucleotide, amino acids, Krebs cycle, and lipid metabolism was observed, consistent with catabolic metabolism. An exception is the accumulation of long fatty acid acylcarnitine for transfer across the inner mitochondrial membrane for β-oxidation. (D) Pathway enrichment analysis supports fatty acid β-oxidation as the primary bioenergetic pathway in stressed cells. Key metabolic pathways, including carnitine biosynthesis and lysine/methionine degradation, contribute to fatty acid β-oxidation. (E) Hypoxia and low glucose induce lysine and methionine degradation to support acylcarnitine biosynthesis and lipid β-oxidation. (F) Joint pathway analysis incorporating transcriptomics and metabolomics studies supports changes from glycolytic to lipolytic metabolism, impacting nucleotides and sugars biosynthesis, including O -GalNAc glycans and protein O -glycosylation. (G and H) Hypoxia and low glucose increase AMP/ATP ratio (G) and activate AMPK by phosphorylation (H), indicating impaired oxidative phosphorylation and potential catabolic processes, including mitophagy. (I) Citrate synthase activity decreases under hypoxia and low glucose, suggesting a reduction in functional mitochondria. (J) TEM analysis reveals major morphological changes, including compromised mitochondria, lipid droplets, peridroplet mitochondria, membrane vesicles, and increased shedding of vesicles, indicating membrane activity changes under stress. Error bars represent mean ± SD for three independent experiments. Mann-Whitney test was used for statistical analysis. Results were considered statistically significant when p < 0.05.

Article Snippet: Human BLCA cell line HT1197 (white male patient) , ATCC , CRL-1473 TM.

Techniques: Membrane, Glycoproteomics, Phospho-proteomics, Activity Assay, Functional Assay, MANN-WHITNEY

Journal: iScience

Article Title: Multilevel plasticity and altered glycosylation drive aggressiveness in hypoxic and glucose-deprived bladder cancer cells

doi: 10.1016/j.isci.2025.111758

Figure Lengend Snippet: Hypoxia and low glucose impair O -glycans extension in BLCA, originating a simple cancer cell glycophenotype (A) BLCA cells exposed to hypoxia and low glucose exhibit less abundant, simpler, and shorter glycomes, lacking extensions beyond core 1 structures. nanoLC-MS/MS analysis shows that this glycophenotype is characterized by sialylated T antigens and core 3, likely due to decreased typical core 1 and 2 structures. DFX-treated cells, stabilizing HIF-1α, show no significant alterations in the glycome, suggesting that changes observed in stressed cells are not driven by HIF-1α. (B) Lectin affinity studies show significant upregulation of Tn and sialylated T antigens (recognized by PNA lectin after Neuraminidase [NeuAse] digestion) under stress, in accordance with MS-based glycomics. Notably, core 3 O -glycans (evaluated by GSL II lectin after PNGase F digestion) remain unchanged, highlighting that cellular stress primarily suppresses core 1/2 O -glycans, rather than increasing core 3 O -glycans. (C) Glucose suppression is the primary driver of glycome remodeling, which can be reversed by reoxygenation and restoration of glucose. (D) Glycogene remodeling is primarily driven by the combined effects of hypoxia and glucose deprivation and leads to a premature halt in glycans extension beyond core 1. C1GALT1C1 , necessary for core 1 biosynthesis, is downregulated, whereas ST3GAL1 , 3 , and 4 are overexpressed, increasing sialylated T antigens and inhibiting core 2 formation. Downregulation of GCNT4 also contributes to core 2 inhibition. Interestingly, elevated GCNT1 and GCNT3 potentially counterbalance core 2 suppression. (E) Quantification of key enzymes involved in O -glycan elongation (C1GalT1; Cosmc; BGnT-6; C2GNT; ST3Gal-I) shows significant upregulation of ST3Gal-1 in stressed cells, consistent with transcriptomics. The others remain unchanged, indicating distinct regulation between glycogenes and glycosyltransferases under these conditions. Bold circles and triangles represent statistically significant changes in T24 and 5637 cell lines, respectively. Error bars represent mean ± SD for three independent experiments. Mann-Whitney Test was used for statistical analysis. Results were considered statistically significant when p < 0.05.

Article Snippet: Human BLCA cell line HT1197 (white male patient) , ATCC , CRL-1473 TM.

Techniques: Tandem Mass Spectroscopy, Inhibition, Glycoproteomics, MANN-WHITNEY

Journal: iScience

Article Title: Multilevel plasticity and altered glycosylation drive aggressiveness in hypoxic and glucose-deprived bladder cancer cells

doi: 10.1016/j.isci.2025.111758

Figure Lengend Snippet: Hypoxic BLCA, characterized by high nuclear HIF-1α expression and low proliferation, shares malignant molecular features with hypoxic and glucose-deprived cells in vitro , including simple glycophenotypes (A and B) Roughly 10% of MIBC tumors display a hypoxic fingerprint (HIF-1α positive /Ki-67 low ) that was not observed in NMIBC and most MIBC tumors (HIF-1α negative /Ki-67 high ), indicating a potential link to aggressiveness. (C) Hypoxic tumors display significantly higher AMPK phosphorylation compared to proliferative cases, denoting a catabolic state. (D) Hypoxic tumors show distinct cellular signaling pathway activation compared to proliferative tumors. PCA for phosphoproteomics data indicates that PC1 (58% variance) primarily separates hypoxic from proliferative tumors, whereas PC2 (15% variance) highlights marked differences among hypoxic tumors. (E) Kinase-Substrate enrichment analysis supports major cell rewiring in hypoxic tumors. Kinases color-coded in red are significantly activated, whereas blue is significantly inactivated. (F) Hypoxic tumors share common kinase activation patterns with stressed BLCA cells in vitro . (G) KEGG pathway enrichment analysis indicates significant alterations in cell signaling pathways, promoting cell motility, cellular senescence, and autophagy in hypoxic tumors as found in stressed cells in vitro . (H) Hypoxic tumors present simple O -glycophenotypes compared to proliferative tumors. nanoLC-MS/MS reveals more homogeneous O -glycome in hypoxic tumors with scarce core 2 glycans. We represent the most abundant structures also found in cell lines, keeping reference to their original relative abundance in relation to all identified glycan species. (I) Hypoxic tumors N -glycome is enriched for oligomannose N -glycans, whereas proliferative tumors are enriched for complex N -glycans. (J and K) Hypoxic tumors show higher levels of Tn and sialylated T antigens and lower levels of sialylated Lewis antigens in O -glycans compared to proliferative tumors, reinforcing the primary suppression of O -glycan extension. NeuAse means sialidase neuraminidase. (L) In hypoxic tumors, Tn and sialylated T antigens co-localize with high HIF-1α. Normoxic, proliferative tumors lack HIF-1α and show low levels of sialylated T antigens and no Tn antigens. Healthy urothelium from non-cancerous individuals served as a negative control for HIF-1α, low Tn, and sialylated T antigens expression. Unpaired t test and Mann-Whitney test were used for statistical analysis. Results were considered statistically significant when p < 0.05.

Article Snippet: Human BLCA cell line HT1197 (white male patient) , ATCC , CRL-1473 TM.

Techniques: Expressing, In Vitro, Phospho-proteomics, Activation Assay, Protein-Protein interactions, Tandem Mass Spectroscopy, Glycoproteomics, Negative Control, MANN-WHITNEY

Journal: iScience

Article Title: Multilevel plasticity and altered glycosylation drive aggressiveness in hypoxic and glucose-deprived bladder cancer cells

doi: 10.1016/j.isci.2025.111758

Figure Lengend Snippet:

Article Snippet: Human BLCA cell line HT1197 (white male patient) , ATCC , CRL-1473 TM.

Techniques: Control, Purification, Recombinant, Plasmid Preparation, Electron Microscopy, Transfection, Staining, Enzyme-linked Immunosorbent Assay, Flow Cytometry, ATP Assay, Phospho-proteomics, Software, Sterility

Journal: BMC Urology

Article Title: Expression patterns of the immune checkpoint ligand CD276 in urothelial carcinoma

doi: 10.1186/s12894-021-00829-0

Figure Lengend Snippet: Cell surface expression of CD276. The density of CD276 on cell surfaces was enumerated by flow cytometry on normal urothelial cells (NUCs) and urothelial cancer cell lines (UCCLs). a The representative NUC (HL19/17) shows a mean fluorescence intensity (MFI) of 2E03 and 91.8% of cells were gated positive for CD276 (solid histogram). b The representative tumor cell line HT1197 shows a MFI of 1.12E04 and 99.1% of cells were gated CD276 positive. Dotted histograms present isotype controls. The x-axis denotes the fluorescence intensity, the Y-axis the number of cells recorded as % of the maximum

Article Snippet: Urothelial cancer cell lines (UCCLs) HT1197 (CRL-1473), TCCsup (HTB-5), RT4 (HTB-2), and 5637 (HTB-9) were purchased from ATCC ( www.atcc.org ).

Techniques: Expressing, Flow Cytometry, Fluorescence

Journal: bioRxiv

Article Title: ARID1A-mutant and deficient bladder cancer is sensitive to EZH2 pharmacologic inhibition

doi: 10.1101/2021.01.12.426383

Figure Lengend Snippet: Bladder cancer cell lines with ARID1A mutation are sensitive to EZH2 inhibition. (A) Immunoblot shows expression of ARID1A in different bladder cancer cell lines including ARID1A mutant (HT1197, HT1376, and VMCUB-1), and ARID1A wild type (T24, 5637, and RT112) cell lines. (B) Immunoblot analysis showing expression level of EZH2, Histone H3 trimethyl lysine 27 (H3K27me3) and total Histone 3 in ARID1A mutant bladder cancer cell lines (HT1197, HT1376 and VM-CUB1) after treatment with EZH2 small molecule inhibitor GSK126. (C) Cell proliferation assay indicated ARID1A mutant bladder cancer cells are sensitive to GSK126. (D) Immunoblot analysis as above in (B) in ARID1A wild type bladder cancer cell lines (T24, 5637 and RT-112) after GSK126 treatment. (E) Cell proliferation assay of ARID1A wild type bladder cancer cells showed no effect of GSK126 treatment. “ns” – non-significant.

Article Snippet: HEK293T (ATCC) and all bladder cancer cell lines HT1197 (ATCC, Manassas, VA, USA), HT1376 (ATCC), T24 (ATCC), 5637 (ATCC), RT112, VM-CUB1 (DSMZ, Braunschweig, Germany) were grown in Dulbecco’s 90% Dulbecco’s MEM (4.5 g/L glucose) with penicillin– streptomycin (100 U/ml) and 10% fetal bovine serum (Sigma-Aldrich, St Louis, MO, USA) in 5% CO 2 cell culture incubator.

Techniques: Mutagenesis, Inhibition, Western Blot, Expressing, Proliferation Assay