Journal: OncoTargets and therapy

Article Title: Nucleolar and Spindle Associated Protein 1 (NUSAP1) Promotes Bladder Cancer Progression Through the TGF-β Signaling Pathway

doi: 10.2147/OTT.S237127

Figure Lengend Snippet: NUSAP1 expression in BLCA tissues and cell lines and survival analysis. ( A ). NUSAP1 mRNA expression levels in 46 BLCA tumors and 14 normal tissues extracted from GSE3167. *** P <0.001 vs Normal. ( B ). Nineteen paired BLCA tumors and normal tissues extracted from the TCGA data portal. *** P <0.001 vs Normal. ( C ). NUSAP1 mRNA expression levels in 25 BLCA tumors and 23 normal tissues obtained from the Department of Urology of the First Affiliated Hospital of Chongqing Medical University. *** P <0.001 vs Normal. ( D and E ). NUSAP1 protein expression in BLCA and normal tissues as detected by immunohistochemistry (100× and 400× magnification). *** P <0.001 vs Normal. ( F ). NUSAP1 protein expression in 6 paired BLCA tissues. ( G ). Survival analysis of patients with BLCA obtained from PrognoScan (GSE13507). ( H and I ). The expression of NUSAP1 in BLCA cell lines was detected by real-time PCR and Western blotting. ** P <0.01, *** P <0.001 vs SV-HUC-1 cells.

Article Snippet: The human BLCA cell lines 5637, T24, BIU87, UM-UC-3, and RT4 and the human ureteric epithelial cell line SV-HUC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Immunohistochemistry, Real-time Polymerase Chain Reaction, Western Blot

Journal: OncoTargets and therapy

Article Title: Nucleolar and Spindle Associated Protein 1 (NUSAP1) Promotes Bladder Cancer Progression Through the TGF-β Signaling Pathway

doi: 10.2147/OTT.S237127

Figure Lengend Snippet: Clinicopathological Characteristics of Patients with BLCA

Article Snippet: The human BLCA cell lines 5637, T24, BIU87, UM-UC-3, and RT4 and the human ureteric epithelial cell line SV-HUC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Biomarker Discovery

Journal: OncoTargets and therapy

Article Title: Nucleolar and Spindle Associated Protein 1 (NUSAP1) Promotes Bladder Cancer Progression Through the TGF-β Signaling Pathway

doi: 10.2147/OTT.S237127

Figure Lengend Snippet: NUSAP1 enhanced the proliferation of BLCA cells. ( A and B ). NUSAP1 expression in transfected 5637 cells determined by PCR and Western blotting. ** P <0.01 vs NC. ( C and D ). NUSAP1 expression in transfected T24 cells by real-time PCR and Western blotting. ** P <0.01 vs NC. ( E and F ). Cell proliferation was determined by a Cell Counting Kit-8 assay. ** P <0.01, *** P <0.001 vs NC (5637) or NC (T24). ( G, H and I ). Cell proliferation was determined by an EdU assay (100× magnification). ** P <0.01 vs NC (5637) or NC (T24).

Article Snippet: The human BLCA cell lines 5637, T24, BIU87, UM-UC-3, and RT4 and the human ureteric epithelial cell line SV-HUC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Transfection, Western Blot, Real-time Polymerase Chain Reaction, Cell Counting, EdU Assay

Journal: OncoTargets and therapy

Article Title: Nucleolar and Spindle Associated Protein 1 (NUSAP1) Promotes Bladder Cancer Progression Through the TGF-β Signaling Pathway

doi: 10.2147/OTT.S237127

Figure Lengend Snippet: NUSAP1 downregulation induced cell cycle arrest and apoptosis in BLCA cells. ( A ). GSEA suggests that NUSAP1 is closely related to the G2/M cell cycle phase. ( B and C ). Cell cycle analysis via flow cytometry. ** P <0.01 vs NC. ( D ). Western blotting analysis of CDK1 and cyclin B1 protein expression. ( E and F ). Apoptosis was analyzed by flow cytometry. ** P <0.01 vs NC (5637), * P <0.05 vs NC (T24). ( G ). Western blotting analysis detected the protein expression levels of Bax, Bcl-2 and cleaved-caspase3.

Article Snippet: The human BLCA cell lines 5637, T24, BIU87, UM-UC-3, and RT4 and the human ureteric epithelial cell line SV-HUC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Cell Cycle Assay, Flow Cytometry, Western Blot, Expressing

Journal: OncoTargets and therapy

Article Title: Nucleolar and Spindle Associated Protein 1 (NUSAP1) Promotes Bladder Cancer Progression Through the TGF-β Signaling Pathway

doi: 10.2147/OTT.S237127

Figure Lengend Snippet: NUSAP1 downregulation in BLCA cells enhanced GEM chemosensitivity and suppressed cell migration, invasion, and EMT. ( A ). The chemosensitivity of BLCA cells to GEM was determined by a Cell Counting Kit-8 assay. * P <0.05, ** P <0.01, *** P <0.001 vs NC (5637) or NC (T24). ( B and C ). A wound healing assay was performed to detect the migration of cells (100× magnification). ** P <0.01, vs NC (5637) or NC (T24). ( D and E ). Transwell assays were performed to evaluate the migration and invasion of cells (100× magnification). ** P <0.01, vs NC (5637) or NC (T24). ( F ). Western blotting analysis detected the protein expression levels of E-cadherin, vimentin and N-cadherin.

Article Snippet: The human BLCA cell lines 5637, T24, BIU87, UM-UC-3, and RT4 and the human ureteric epithelial cell line SV-HUC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Migration, Cell Counting, Wound Healing Assay, Western Blot, Expressing

Journal: OncoTargets and therapy

Article Title: Nucleolar and Spindle Associated Protein 1 (NUSAP1) Promotes Bladder Cancer Progression Through the TGF-β Signaling Pathway

doi: 10.2147/OTT.S237127

Figure Lengend Snippet: NUSAP1 regulated the epithelial-mesenchymal transition of BLCA via the TGF-β signaling pathway. ( A ). GSEA revealed that NUSAP1 expression was positively correlated with the TGFBR complex and the Smad2/3 pathway in BLCA. ( B ). Gene correlation analysis of NUSAP1 and TGFBR1 obtained from Gene Expression Profiling Interactive Analysis (GEPIA, http://gepia.cancer-pku.cn/ ). *** P <0.001. ( C ). Real-time PCR was performed to detect the relationship between NUSAP1 and TGFBR1. ** P <0.01, vs NC (5637) or NC (T24). ( D ). Western blotting analysis of the protein expression levels of TGFBR1, Smad2/3, p- Smad2/3, E-cadherin, and vimentin. ( E and F ). Wound healing assays were performed to detect the migration of cells (100× magnification) * P <0.05, ** P <0.01. ( G and H ). Transwell assays were performed to evaluate the migration and invasion of cells (100× magnification). * P <0.05, ** P <0.01, *** P <0.001. ( I ). Western blotting analysis of the protein expression levels of Smad2/3, p- Smad2/3, E-cadherin, and vimentin.

Article Snippet: The human BLCA cell lines 5637, T24, BIU87, UM-UC-3, and RT4 and the human ureteric epithelial cell line SV-HUC-1 were obtained from the American Type Culture Collection (ATCC, Manassas, VA, USA).

Techniques: Expressing, Gene Expression, Real-time Polymerase Chain Reaction, Western Blot, Migration

Journal: Journal of Translational Medicine

Article Title: TRIM38 triggers the uniquitination and degradation of glucose transporter type 1 (GLUT1) to restrict tumor progression in bladder cancer

doi: 10.1186/s12967-021-03173-x

Figure Lengend Snippet: TRIM38 inhibition enhanced BLCA proliferation, migration and stemness features. a Western blot assay exhibiting the knockout efficiency of TRIM38 in T24 and EJ cells. b Western blot assay showing the overexpression of TRIM38 in T24 and EJ cells. c Colony formation capacity was determined in Ctrl and TRIM38-KO cells (T24 and EJ). Quantification of results were showed on the right panel. d CCK-8 assays were conducted to exhibit the cell growth in the indicated cells (T24, EJ, 5637) at every timepoints. e Transwell assays showed that the invasive capacity of BLCA cells was enhanced with TRIM38 ablation (left panel). Quantification of results was exhibited on the right panel. Scale bars = 250 μm. f The invasive ability of EJ cells was suppressed when cells were transfected with TRIM38 (left panel). Quantification of results was exhibited on the right panel. Scale bars = 250 μm. g Sphere formation assay was conducted to confirm that TRIM38 deficiency could notably drive stemness features of T24 cells. Quantification of results was exhibited on the right panel. Scale bars = 200 μm

Article Snippet: The human BLCA cell lines (T24, EJ and 5637) and 293 T cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Inhibition, Migration, Western Blot, Knock-Out, Over Expression, CCK-8 Assay, Transfection, Tube Formation Assay

Journal: Journal of Translational Medicine

Article Title: TRIM38 triggers the uniquitination and degradation of glucose transporter type 1 (GLUT1) to restrict tumor progression in bladder cancer

doi: 10.1186/s12967-021-03173-x

Figure Lengend Snippet: GLUT1 inhibitor (BAY-876) was effective to inhibit the progression of TRIM38 low/− bladder cancer. a The half maximal inhibitory concentration (IC50) values of BAY-876 (GLUT1 inhibitor) in BLCA cell lines (T24, EJ and 5637). b – d BAY-876 could suppress the cell growth of BLCA cells in a dose-dependent manner. e Representative image inhibited that BAY-876 could inhibit the tumor volumes derived from TRIM38-KO cells relative to tumors derived from control cells. f The tumor volumes were detected and recorded at the indicated timepoints and the growth curve was generated. g Tumors were resected and weighed to compare the differences in the indicated groups

Article Snippet: The human BLCA cell lines (T24, EJ and 5637) and 293 T cells were obtained from the American Type Culture Collection (ATCC).

Techniques: Concentration Assay, Derivative Assay, Control, Generated

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 5637 (white male patient) , ATCC , HTB-9TM.

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 5637 (white male patient) , ATCC , HTB-9TM.

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 5637 (white male patient) , ATCC , HTB-9TM.

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 5637 (white male patient) , ATCC , HTB-9TM.

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 5637 (white male patient) , ATCC , HTB-9TM.

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 5637 (white male patient) , ATCC , HTB-9TM.

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