Journal: International Journal of Molecular Sciences

Article Title: Galactokinase 1 Inhibition-Induced Cell Cycle Arrest and Apoptosis in Bladder Cancer Cells Is Associated with AKT Signaling Downregulation

doi: 10.3390/ijms27062911

Figure Lengend Snippet: GALK1 knockdown decreased cell proliferation and inhibited the migration and invasion of BCa cells. ( a ) Microscopic image of the MB49 cells with knockdown of GALK1 expression using shRNA. ( b ) Trypan blue assay showing cell viability after GALK1 knockdown at different time points in MB49 cells. ( c ) Western blot analysis was performed to assess the levels of GALK1, PCNA, and β-actin in GALK1 knockdown MB49 cells. ( d ) Transwell migration and invasion assays were conducted to evaluate the migratory and invasive potential of BCa cells after GALK1 knockdown. ( e ) Representative OCT images and 3D volume renderings of MB49 cells-derived spheroids after 3 days of growth. Data shown are mean ± SEM of triplicate ( n = 3) samples for each treatment. ** p < 0.01, and ‘ns’ for no significance.

Article Snippet: MB49 mouse BCa cells were obtained from Accegen, Fairfield, NJ, USA (ABC-TC2235S) and cultured in DMEM (ATCC # 30-2002) media.

Techniques: Knockdown, Migration, Expressing, shRNA, Western Blot, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Galactokinase 1 Inhibition-Induced Cell Cycle Arrest and Apoptosis in Bladder Cancer Cells Is Associated with AKT Signaling Downregulation

doi: 10.3390/ijms27062911

Figure Lengend Snippet: GALK1 inhibitor Cpd36 inhibits the proliferation, migration, and invasion of BCa cells. ( a ) Chemical structure of GALK1 inhibitor Cpd36. ( b ) MTT assay was used to assess the growth-inhibitory effect of Cpd36 on BCa cells after 48 h of treatment. Red dotted line represents IC50. ( c ) 5637, SCaBER, and MB49 cells were treated with 20–80 µg∕ml for 48 h, and the percentage of cell viability was calculated using trypan blue dye. ( d ) Western blot analysis was performed to examine the protein expression of GALK1 and PCNA in BCa cells. ( e ) Representative image showing the Transwell migration and invasion assay of BCa cells after 48 h of Cpd36 treatment. ( f ) Western blot analysis of MMP-9 and β-actin in BCa cells. ( g ) Effect of Cpd36 on UPII-SV40T tumor-derived organoids and BBN-rat tumor-derived BCa organoids, magnification 20×; Scale bar, 200 µm. The results are representative of three replicates ( n = 3) mean ± SEM. Significance is indicated by * p < 0.05, ** p < 0.01, *** p < 0.001, and ‘ns’ for no significance.

Article Snippet: MB49 mouse BCa cells were obtained from Accegen, Fairfield, NJ, USA (ABC-TC2235S) and cultured in DMEM (ATCC # 30-2002) media.

Techniques: Migration, MTT Assay, Western Blot, Expressing, Invasion Assay, Derivative Assay

Journal: International Journal of Molecular Sciences

Article Title: Galactokinase 1 Inhibition-Induced Cell Cycle Arrest and Apoptosis in Bladder Cancer Cells Is Associated with AKT Signaling Downregulation

doi: 10.3390/ijms27062911

Figure Lengend Snippet: Cpd36 arrests the cell cycle and induces apoptosis in BCa cells. ( a ) Flow cytometry analysis showing cell cycle progression in 5637, SCaBER, and MB49 cells treated with Cpd36 for 48 h. The cell populations in the G1, S, and G2/M phases were determined. ( b ) Western blot analysis was performed to assess Cyclin D1, p21, and p27 expression, with the membranes reprobed for β-actin to verify equal protein loading. ( c ) Annexin V staining was performed to detect the induction of apoptosis in BCa cells. ( d ) Western blot analysis was performed to detect the apoptosis markers cleaved-PARP and cleaved-caspase-3 in BCa cells. The densitometric value for each protein is quantified using ImageJ software 1.54r and is displayed below each band as the fold change relative to the control. ** p < 0.01, *** p < 0.001, and ‘ns’ for no significance.

Article Snippet: MB49 mouse BCa cells were obtained from Accegen, Fairfield, NJ, USA (ABC-TC2235S) and cultured in DMEM (ATCC # 30-2002) media.

Techniques: Flow Cytometry, Western Blot, Expressing, Staining, Software, Control

Journal: International Journal of Molecular Sciences

Article Title: Galactokinase 1 Inhibition-Induced Cell Cycle Arrest and Apoptosis in Bladder Cancer Cells Is Associated with AKT Signaling Downregulation

doi: 10.3390/ijms27062911

Figure Lengend Snippet: Cpd36 decreased the mitochondrial membrane potential and induced mitochondrial ROS production in BCa cells. ( a ) JC-1 staining was used to assess the mitochondrial membrane potential in 5637, SCaBER, and MB49 cells treated with Cpd36 or CCCP (as a positive control) for 48 h. ( b ) The fluorescence intensity of mitochondrial O2– was measured using MitoSOX TM Red after 48 h of Cpd36 treatment. Data are presented as mean ± SEM, with significance levels indicated as * p < 0.05, ** p < 0.01, *** p < 0.001, and ns for no significance.

Article Snippet: MB49 mouse BCa cells were obtained from Accegen, Fairfield, NJ, USA (ABC-TC2235S) and cultured in DMEM (ATCC # 30-2002) media.

Techniques: Membrane, Staining, Positive Control, Fluorescence

Journal: International Journal of Molecular Sciences

Article Title: Galactokinase 1 Inhibition-Induced Cell Cycle Arrest and Apoptosis in Bladder Cancer Cells Is Associated with AKT Signaling Downregulation

doi: 10.3390/ijms27062911

Figure Lengend Snippet: Cpd36 effects Akt signaling in BCa cells. Western blot analysis of p-AKT, AKT, and β-actin protein levels in 5637, SCaBER, and MB49 cells after treatment with Cpd36 for 48 h using appropriate primary and secondary antibodies. Western blot data are shown as mean ± SEM from two replicates ( n = 2). * p < 0.05.

Article Snippet: MB49 mouse BCa cells were obtained from Accegen, Fairfield, NJ, USA (ABC-TC2235S) and cultured in DMEM (ATCC # 30-2002) media.

Techniques: Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Galactokinase 1 Inhibition-Induced Cell Cycle Arrest and Apoptosis in Bladder Cancer Cells Is Associated with AKT Signaling Downregulation

doi: 10.3390/ijms27062911

Figure Lengend Snippet: Cpd36 treatment sensitized BCa cells to chemotherapy drugs. 5637, SCaBER, and MB49 cells were incubated with Cpd36, or its combination with cisplatin ( a ) or gemcitabine ( b ), for 48 h. Cell viability was determined using the MTT assay. Combination index values were calculated and are mentioned in the graph for the combination of Cpd36 and cisplatin or gemcitabine. Data is shown as the mean± SEM from three independent experiments.

Article Snippet: MB49 mouse BCa cells were obtained from Accegen, Fairfield, NJ, USA (ABC-TC2235S) and cultured in DMEM (ATCC # 30-2002) media.

Techniques: Incubation, MTT Assay

Journal: Oncoimmunology

Article Title: Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models

doi: 10.1080/2162402X.2024.2370544

Figure Lengend Snippet: In vivo therapeutic efficacy of CD25-targeted NIR-DPR in a MB49-luc tumor mouse model.

Article Snippet: HT2-A5E and EL4 were purchased from ATCC (Manassas, VA, USA), MB49-luc and Pan02-luc from GenTarget Inc (San Diego, CA, USA), MOC1 and MOC2 from Kerafast (Boston, MA, USA), and LL/2-luc from Imanis Life Sciences (Rochester, MN, USA).

Techniques: In Vivo

Journal: Oncoimmunology

Article Title: Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models

doi: 10.1080/2162402X.2024.2370544

Figure Lengend Snippet: Early host immune responses to CD25-targeted NIR-DPR combined with PD-1 blockade in a MB49-luc tumor mouse model.

Article Snippet: HT2-A5E and EL4 were purchased from ATCC (Manassas, VA, USA), MB49-luc and Pan02-luc from GenTarget Inc (San Diego, CA, USA), MOC1 and MOC2 from Kerafast (Boston, MA, USA), and LL/2-luc from Imanis Life Sciences (Rochester, MN, USA).

Techniques:

Journal: Oncoimmunology

Article Title: Near-infrared duocarmycin photorelease from a Treg-targeted antibody-drug conjugate improves efficacy of PD-1 blockade in syngeneic murine tumor models

doi: 10.1080/2162402X.2024.2370544

Figure Lengend Snippet: Enhanced anti-cancer immunity was successfully established after CD25-targeted NIR-DPR combined with PD-1 blockade in a MB49-luc tumor mouse model.

Article Snippet: HT2-A5E and EL4 were purchased from ATCC (Manassas, VA, USA), MB49-luc and Pan02-luc from GenTarget Inc (San Diego, CA, USA), MOC1 and MOC2 from Kerafast (Boston, MA, USA), and LL/2-luc from Imanis Life Sciences (Rochester, MN, USA).

Techniques:

Journal: Journal of Clinical Medicine

Article Title: Nuclear Factor-κB Overexpression is Correlated with Poor Outcomes after Multimodality Bladder-Preserving Therapy in Patients with Muscle-Invasive Bladder Cancer

doi: 10.3390/jcm8111954

Figure Lengend Snippet: NFκB signaling was up-regulated after irradiation (RT) and inhibited with the treatment of an NFκB inhibitor, SC75741, mitigating the proliferative capability of bladder cancer cells. ( A ) Images and quantification by densitometry and ImageJ of Western blots for NFκB transcription factor proteins (p65 and p50) (n = 3) (n = 3) and ( B ) colony formation assays of MB49 and T24 cancer cells after pre-treatment with or without SC75741 (5 μM) and/or irradiation (5 Gy) (n = 4). * p < 0.05.

Article Snippet: Human bladder urothelial carcinoma cell line, T24, and murine bladder tumor cell line, MB49, were purchased from the ATCC/Bioresource Collection and Research Center (BCRC).

Techniques: Irradiation, Western Blot

Journal: Journal of Clinical Medicine

Article Title: Nuclear Factor-κB Overexpression is Correlated with Poor Outcomes after Multimodality Bladder-Preserving Therapy in Patients with Muscle-Invasive Bladder Cancer

doi: 10.3390/jcm8111954

Figure Lengend Snippet: Blockade of NFκB signaling by RNA interference inhibited radiation (RT)-induced nuclear translocation of phosphor-p65 and the invasiveness capability of murine bladder cancer cells. ( A ) Immunofluorescence staining of phospho-p65 at 0, 1, and 3 h after RT (5 Gy) of MB49 vector-control (Ctrl) and MB49 NFκB knock-down (KD) cells. ( B ) MB49 control (Ctrl) and MB49 NFκB KD cells were seeded in Matrigel-coated inserts of Boyden chambers, treated without and with RT (5 Gy). After 24 h, the invading cells were viewed microscopically (high-power field, 200 ×, left panel). Invading cells were counted. * p < 0.05, Ctrl sham group vs. Ctrl RT group.

Article Snippet: Human bladder urothelial carcinoma cell line, T24, and murine bladder tumor cell line, MB49, were purchased from the ATCC/Bioresource Collection and Research Center (BCRC).

Techniques: Translocation Assay, Immunofluorescence, Staining, Plasmid Preparation, Control, Knockdown