Journal: Frontiers in Immunology

Article Title: ATP13A2 promoted cell stemness, cisplatin resistance, autophagy, and cell progression of bladder cancer

doi: 10.3389/fimmu.2026.1743064

Figure Lengend Snippet: Knockdown of ATP13A2 inhibited cell activity, migration, and invasion and promoted cell apoptosis in bladder cancer. (A) QPCR analysis of ATP13A2 mRNA expression in T24 cells transfected with sh-NC or sh-ATP13A2. (B) WB analysis of ATP13A2 protein levels in transfected cells. (C) Colony formation assay to evaluate cell proliferation. (D) Transwell assays to assess cell migration and invasion in transfected cells. (E) Wound-healing assay to measure cell migration rate. (F) WB analysis of apoptosis-related proteins (e.g., Cleaved caspase-3, Bax, Bcl-2). (G) Flow cytometry to analyze apoptosis ratio in transfected cells. N = 3, data are presented as mean ± SD. Statistical significance between the two groups was determined by unpaired Student’s t-test. * P < 0.05, ** P < 0.01, **** P < 0.0001.

Article Snippet: Human bladder cancer T24 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Knockdown, Activity Assay, Migration, Expressing, Transfection, Colony Assay, Wound Healing Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: ATP13A2 promoted cell stemness, cisplatin resistance, autophagy, and cell progression of bladder cancer

doi: 10.3389/fimmu.2026.1743064

Figure Lengend Snippet: Knockdown of ATP13A2 inhibited autophagy, decreased stemness, and drug resistance. (A) Tumor spheroid growth was performed to assess the cell stemness in T24 cells transfected with sh-NC or sh-ATP13A2. (B) The autophagy biomarkers of LC3 and p62 were detected by immunofluorescence in transfected cells. (C) CCK-8 assay determined the cell activity under graded cisplatin treatments (0, 10, 50, 100, and 200 μg/mL). (D) Colony formation was conducted to detect the cell activity under cisplatin treatment at an IC25 concentration in transfected cells. (E) Flow cytometry assessed the cell apoptosis under cisplatin treatment at an IC 25 concentration in transfected cells. N = 3, data are presented as mean ± SD. Statistical significance between the two groups was determined by unpaired student’s t-test. *** P < 0.001, **** P < 0.0001.

Article Snippet: Human bladder cancer T24 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Knockdown, Transfection, Immunofluorescence, CCK-8 Assay, Activity Assay, Concentration Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: ATP13A2 promoted cell stemness, cisplatin resistance, autophagy, and cell progression of bladder cancer

doi: 10.3389/fimmu.2026.1743064

Figure Lengend Snippet: Overexpressed ATP13A2 promoted cell stemness, cisplatin resistance, and autophagy. (A) QPCR analysis determined the mRNA expression level of ATP13A2 in T24 cells transfected with vector or ATP13A2 overexpression plasmid. (B) WB analysis evaluated the protein expression level of ATP13A2 in transfected cells. (C) Tumor spheroid growth was performed to assess the cell stemness in transfected cells. (D) The autophagy biomarkers of LC3 and p62 were detected by immunofluorescence in transfected cells. (E) CCK-8 assay determined the cell activity under graded cisplatin treatments (0, 10, 50, 100, and 200μg/mL). (F) Colony formation was conducted to detect the cell activity under cisplatin treatment at an IC 25 concentration in transfected cells. (G) Flow cytometry assessed the cell apoptosis under cisplatin treatment at an IC 25 concentration in transfected cells. N = 3, data are presented as mean ± SD. Statistical significance between the two groups was determined by unpaired student’s t-test. ** P < 0.01.

Article Snippet: Human bladder cancer T24 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Expressing, Transfection, Plasmid Preparation, Over Expression, Immunofluorescence, CCK-8 Assay, Activity Assay, Concentration Assay, Flow Cytometry

Journal: Frontiers in Immunology

Article Title: ATP13A2 promoted cell stemness, cisplatin resistance, autophagy, and cell progression of bladder cancer

doi: 10.3389/fimmu.2026.1743064

Figure Lengend Snippet: Autophagy inhibitors inhibited cell activity, migration, and invasion and promoted apoptosis of bladder cancer cells. (A) Colony formation was performed to evaluate cell proliferation in T24 cells treated with bafilomycin A1. (B) Transwell assays assessed the cell migration and invasion in treated cells. (C) Wound-healing assay measured the cell migration rate in treated cells. (D) The cell apoptosis ratio was observed in treated cells by flow cytometry. (E) WB analysis was performed to assess the expression levels of apoptosis-related proteins in cells treated with bafilomycin A1. N = 3, data are presented as mean ± SD. Statistical significance between the two groups was determined by unpaired student’s t-test. * P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001.

Article Snippet: Human bladder cancer T24 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Activity Assay, Migration, Wound Healing Assay, Flow Cytometry, Expressing

Journal: Frontiers in Immunology

Article Title: ATP13A2 promoted cell stemness, cisplatin resistance, autophagy, and cell progression of bladder cancer

doi: 10.3389/fimmu.2026.1743064

Figure Lengend Snippet: Autophagy inhibitor can significantly inhibit cell stemness and weaken drug resistance. (A) Tumor spheroid growth was performed to assess the cell stemness in T24 cells treated with bafilomycin A1. (B) The autophagy biomarkers of LC3 and p62 were detected by immunofluorescence (DAPI was used for nuclear staining) in treated cells. (C) CCK-8 assay determined the cell activity under graded cisplatin treatments (0, 10, 50, 100, and 200μg/mL). (D) Colony formation was conducted to detect the cell activity under cisplatin treatment at an IC25 concentration in treated cells. (E) Flow cytometry assessed the cell apoptosis under cisplatin treatment at an IC 25 concentration in treated cells. N = 3, data are presented as mean ± SD. Statistical significance between the two groups was determined by unpaired student’s t-test. *** P < 0.001, **** P < 0.0001.

Article Snippet: Human bladder cancer T24 cells were obtained from the American Type Culture Collection (Manassas, VA, USA) and cultured in high-glucose DMEM supplemented with 10% fetal bovine serum (FBS, Gibco, USA) and 1% penicillin-streptomycin at 37°C in 5% CO 2 .

Techniques: Immunofluorescence, Staining, CCK-8 Assay, Activity Assay, Concentration Assay, Flow Cytometry

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Betulinic acid (BA) enhances tumor necrosis factor (TNF)-related apoptosis-inducing ligand (TRAIL)-mediated cytotoxicity in T24 human bladder cancer cells. T24 cells were treated with the indicated concentrations of BA and TRAIL alone or in combination for 24 h. (A, B) After treatment, cell viability was assessed via 3′-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT) assay. (C) Morphological changes in the cells treated with BA and TRAIL alone or in combination were observed under an inverted microscope. (D, E) After 4′,6-diamidino-2-phenylindole (DAPI) staining, nuclear morphological changes were observed under a fluorescence microscope (D), and the frequency of cells with chromatin condensation and fragmentation was determined (E). (F, G) Flow cytometry was performed after propidium iodide (PI) staining. Representative histograms (F) and the frequency of cells in the sub-G1 phase (G) are shown. * p <0.05, ** p <0.01, and *** p <0.001 vs. control cells; ### p <0.001 vs. BA-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: MTT Assay, Inverted Microscopy, Staining, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL increases reactive oxygen species (ROS) production and decreases ATP levels in T24 human bladder cancer cells. T24 cells were treated with BA and TRAIL alone or in combination and cultured for 30 min (A-C) or 24 h (D). (A, B) After treatment, the cells were stained with 2′,7′-dichlorofluorescein diacetate (DCF-DA) and analyzed via flow cytometry. Representative histograms (A) and the frequency of DCF-positive cells (B) are shown. (C) Representative fluorescence images of the cells stained with DCF-DA, indicating ROS production, were captured via fluorescence microscopy. (D) Intracellular ATP levels were measured using an ATP assay kit. * p <0.05 and ** p <0.01 vs. control cells; ### p <0.001 vs. BA-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Cell Culture, Staining, Flow Cytometry, Fluorescence, Microscopy, ATP Assay, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL increases mitochondrial damage and induces changes in the expression levels of Bcl-2 family proteins in T24 human bladder cancer cells. T24 cells were treated with BA and TRAIL alone or in combination and cultured for 24 h. (A, B) After treatment, the cells were stained with 5,5′,6,6′-tetrachloro-1,1′,3,3′-tetramethylbenzimidazolyl carbocyanine iodide (JC-1) and analyzed via flow cytometry. Representative histograms (A) and the frequency of cells with JC-1 monomers (B), indicating mitochondrial membrane potential (MMP) loss, are shown. * p <0.05 vs. control cells; ### p <0.001 vs. BA-treated cells (n=3). (C, D) Mitochondrial and cytosolic fractions were isolated from the cells (C), total protein was extracted (D), and immunoblotting was performed using antibodies against target proteins. Cytochrome c oxidase (COX IV) and β-actin were used as loading controls for the two fractions, respectively.

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Expressing, Cell Culture, Staining, Flow Cytometry, Membrane, Control, Isolation, Western Blot

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL activates the caspase-dependent extrinsic and intrinsic apoptotic pathways in T24 human bladder cancer cells. T24 cells were either directly co-treated with BA and TRAIL or pretreated with z-VAD-fmk for 1 h before co-treatment with BA and TRAIL and cultured for 24 h. (A) Total protein was extracted, and immunoblotting analysis was performed using antibodies against target proteins. (B) Caspase activity was examined using caspase assay kits. (C, D) After DAPI staining, nuclear morphological changes were observed under a fluorescence microscope (C), and the frequency of cells with chromatin condensation and fragmentation was determined (D). (E, F) Flow cytometry was performed after PI staining. Representative histograms (E) and the frequency of cells in the sub-G1 phase (F) are shown. ** p <0.01 and *** p <0.001 vs. control cells; ### p <0.001 vs. BA and TRAIL co-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Cell Culture, Western Blot, Activity Assay, Caspase Assay, Staining, Fluorescence, Microscopy, Flow Cytometry, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL inhibits activation of the phosphoinositide 3-kinase (PI3K)/Akt pathway in T24 human bladder cancer cells. T24 cells were either directly co-treated with BA and TRAIL or pretreated with LY294002 for 1 h before co-treatment with BA and TRAIL and cultured for 24 h. (A) Total cellular proteins were isolated from the cells, and phosphorylation levels of PI3K and Akt were determined via immunoblotting. (B, C) After DAPI staining, nuclear morphological changes were observed under a fluorescence microscope (B), and the frequency of cells with chromatin condensation and fragmentation was determined (C). (D, E) Flow cytometry was performed after PI staining. Representative histograms (D) and the frequency of cells in the sub-G1 phase (E) are shown. (F) Cell viability was assessed via MTT assay. *** p <0.001 vs. control cells; ### p <0.001 vs. BA and TRAIL co-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Activation Assay, Cell Culture, Isolation, Phospho-proteomics, Western Blot, Staining, Fluorescence, Microscopy, Flow Cytometry, MTT Assay, Control

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Combination treatment with BA and TRAIL increases apoptosis in T24 human bladder cancer cells in an ROS-dependent manner. T24 cells were pretreated with N-acetyl-l-cysteine (NAC) 1 h before combination treatment with BA and TRAIL and cultured for 24 h. (A, B) Total protein was extracted and analyzed via immunoblotting using antibodies against target proteins. β-actin was used as a loading control. (C) Caspase-3 activity was measured using a caspase-3 assay kit. (D, E) After DAPI staining, nuclear morphological changes were observed under a fluorescence microscope (D), and the frequency of cells with chromatin condensation and fragmentation was determined (E). (F, G) Flow cytometry was performed after PI staining. Representative histograms (F) and the frequency of cells in the sub-G1 phase (G) are shown. (H) Cell viability was assessed via MTT assay. *** p <0.001 vs. control cells; ### p <0.001 vs. BA and TRAIL co-treated cells (n=3).

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Cell Culture, Western Blot, Control, Activity Assay, Caspase-3 Assay, Staining, Fluorescence, Microscopy, Flow Cytometry, MTT Assay

Journal: Biomolecules & Therapeutics

Article Title: Combination Therapy with Betulinic Acid and TRAIL Increases ROS-Dependent Cytotoxicity and Inhibits PI3K/Akt Signaling in Human Bladder Cancer Cells

doi: 10.4062/biomolther.2026.035

Figure Lengend Snippet: Schematic diagram of cytotoxicity induction in betulinic acid and TRAIL co-treated T24 human bladder cancer cells. TRAIL, tumor necrosis factor-related apoptosis-inducing ligand; PI3K, phosphoinositide 3-kinase; NAC, N-acetyl-l-cysteine; MMP, mitochondrial membrane potential; FADD, Fas-associated death domain; tBid, truncation of BH3-interacting domain death agonist; PARP, poly(ADP-ribose) polymerase.

Article Snippet: T24 human bladder cancer cells obtained from the American Type Culture Collection (Manassas, VA, USA) were cultured as previously described ( Kim et al. , 2021 ).

Techniques: Membrane

Journal: Biomedicines

Article Title: Time-Dependent Effects of Cisplatin on Autophagy Gene Expression in Bladder Cancer Cells

doi: 10.3390/biomedicines14051006

Figure Lengend Snippet: Time-dependent cytotoxic effect of cisplatin and determination of IC 50 values in T24 bladder cancer cells. Cell viability was evaluated at 24 and 48 h time points following exposure to increasing concentrations of cisplatin (0–40 µM). The resulting dose–response curves demonstrate that cisplatin decreases cell viability in a dose- and time-dependent manner. The horizontal dashed line represents 50% cell viability, while the vertical dashed lines indicate the corresponding IC 50 values at the respective time points. The lower IC 50 values observed at 48 h of exposure indicate the increasing cytotoxic effect of cisplatin over time.

Article Snippet: Human bladder cancer cell lines T24 and 5637 (ATCC, Manassas, VA, USA) were utilized in this study.

Techniques:

Journal: Biomedicines

Article Title: Time-Dependent Effects of Cisplatin on Autophagy Gene Expression in Bladder Cancer Cells

doi: 10.3390/biomedicines14051006

Figure Lengend Snippet: Time-dependent cytotoxic effect of cisplatin and determination of IC 50 values in 5637 bladder cancer cells. Cells were exposed to varying concentrations of cisplatin for 24 and 48 h, and cell viability was assessed. The dose–response curves indicate a more pronounced sensitivity to cisplatin in 5637 cells. The horizontal dashed line represents 50% cell viability, while the vertical dashed lines indicate the calculated IC 50 values. Lower IC 50 values compared to T24 cells demonstrate that this cell line is more sensitive.

Article Snippet: Human bladder cancer cell lines T24 and 5637 (ATCC, Manassas, VA, USA) were utilized in this study.

Techniques:

Journal: Biomedicines

Article Title: Time-Dependent Effects of Cisplatin on Autophagy Gene Expression in Bladder Cancer Cells

doi: 10.3390/biomedicines14051006

Figure Lengend Snippet: Time-dependent heatmap of autophagy-related gene expression in T24 and 5637 bladder cancer cells following cisplatin treatment. Relative expression levels (2 −ΔΔCt ) are shown for genes involved in autophagy initiation, elongation, selective autophagy, and lysosomal function across 0, 6, 24, and 48 h.

Article Snippet: Human bladder cancer cell lines T24 and 5637 (ATCC, Manassas, VA, USA) were utilized in this study.

Techniques: Gene Expression, Expressing

Journal: Biomedicines

Article Title: Time-Dependent Effects of Cisplatin on Autophagy Gene Expression in Bladder Cancer Cells

doi: 10.3390/biomedicines14051006

Figure Lengend Snippet: Time-dependent expression profiles of autophagy-related genes in T24 and 5637 bladder cancer cell lines following cisplatin treatment. Relative mRNA expression levels of genes involved in autophagy initiation ( ULK1 , BECN1 ), elongation ( ATG5 , ATG7 , LC3B ), selective autophagy ( p62/SQSTM1 ), and lysosomal regulation (LAMP1 , TFEB ) were evaluated at 0, 6, 24, and 48 h post-treatment. Gene expression levels were calculated using the 2 −ΔΔCt method and are presented as fold changes relative to the control group (0 h). Blue bars represent the T24 cell line, and orange bars represent the 5637 cell line. Data are expressed as mean ± SEM of at least three independent experiments. * p < 0.05, ** p < 0.01 indicating statistical significance between the T24 and 5637 cell lines at the corresponding time points.

Article Snippet: Human bladder cancer cell lines T24 and 5637 (ATCC, Manassas, VA, USA) were utilized in this study.

Techniques: Expressing, Gene Expression, Control