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: Cancer Research

Article Title: Adipocyte Precursor-Derived NRG1 Promotes Resistance to FGFR Inhibition in Urothelial Carcinoma

doi: 10.1158/0008-5472.CAN-23-1398

Figure Lengend Snippet: Adipocyte precursors promote resistance against erdafitinib in bladder cancer cell lines. A, Western blot analysis of FGFR3 in four bladder cancer cell lines: RT4, RT112, TSCCUP, and T24. β-Actin served as a loading control. B, Proliferation analysis of five bladder cancer cell lines RT4, RT112, TSCCUP, T24, and MB49 treated with vehicle (Veh) or erdafitinib (1, 10, 100, 1,000 nmol/L). Crystal violet staining was done on day 7. Three biological replicates were performed. Data are represented as mean ± SD. C, Western blot analysis of pAKT and pERK1/2 in RT4 and RT112 cells treated with DMSO (Veh) or erdafitinib (10 and 100 nmol/L) for 16 hours. β-Actin served as a loading control. D, Schematic diagram of the proliferation assay performed to investigate the effect of CM of different stromal cells on erdafitinib response. E, Proliferation analysis of RT4 and RT112 cells treated with 10 nmol/L erdafitinib in media control (ctrl) or CM of 3T3-L1 cells or hADSC. Crystal violet staining was performed on day 7. Data are normalized to cells treated with vehicle. Four biological replicates were performed. Data are represented as mean ± SD. F, Western blot analysis of pAKT and pERK1/2 in RT4 and RT112 cells treated with 10 nmol/L erdafitinib in media control or CM of 3T3-L1 cells. β-Actin served as a loading control. G, Proliferation analysis of RT4 cells and RT112 cells treated with 10 nmol/L erdafitinib in CM of 3T3-L1 cells or heat-inactivated (HI) CM of 3T3-L1. Data were normalized to cells treated with vehicle. Crystal violet staining was performed on day 7. Three biological replicates were performed. Data are represented as mean ± SD. Two-way ANOVA was used for statistical analysis. *, P < 0.05; **, P < 0.01; ***, P < 0.001; ****, P < 0.0001.

Article Snippet: MB49 cell line was purchased from Addexbio, RT112 cell line was purchased from DSMZ (German Collection of Microorganisms and Cell Cultures), and human adipose-derived stem cells (hADSC) were purchased from Lonza.

Techniques: Western Blot, Staining, Proliferation 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: International Journal of Molecular Sciences

Article Title: Combination of Cisplatin and Irradiation Induces Immunogenic Cell Death and Potentiates Postirradiation Anti–PD-1 Treatment Efficacy in Urothelial Carcinoma

doi: 10.3390/ijms22020535

Figure Lengend Snippet: Concurrent anti–PD-1 treatment facilitated abscopal effects induced by a single dose of irradiation in MB49 tumor-bearing mice. ( A ) Scheme for tumor inoculation and treatments. Six- to seven-week-old mice are inoculated subcutaneously with MB49 cells in the left hindlimb and in the right flank, and seven days later, receive irradiation with a single fraction of 10 Gy to the left hindlimb and anti–PD-1 treatment (or its isotype controls). ( B ) MB49 tumor growth curves of irradiated tumors in the left hindlimb (left) and nonirradiated tumors in the right flank (right). ( C ) Survival curves of mice. Log-rank test is used to compare survival curves. All data are shown as the mean ± standard error of the mean (SEM). Asterisks indicate p -values comparing two groups, as indicated in the figure. * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: MB49, a mouse bladder UC cell line, was purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Irradiation

Journal: International Journal of Molecular Sciences

Article Title: Combination of Cisplatin and Irradiation Induces Immunogenic Cell Death and Potentiates Postirradiation Anti–PD-1 Treatment Efficacy in Urothelial Carcinoma

doi: 10.3390/ijms22020535

Figure Lengend Snippet: The combination of cisplatin and irradiation potentiated the efficacy of postirradiation anti–PD-1 treatment in MB49 tumor-bearing mice. ( A ) Scheme for tumor inoculation, treatments, and T cell analysis. For the CRT model (top), six- to seven-week-old mice are injected with MB49 cells in the left hindlimb, and seven days later, receive cisplatin at 3 mg/kg and irradiation with a single fraction of 10 Gy to the left hindlimb. The mice are injected with MB49 cells in the right flank 14 days after irradiation, and seven days later, receive anti–PD-1 treatment (or its isotype controls). For the Non-CRT model (bottom), only a right flank tumor is established in the mice, and cisplatin and irradiation are not given. ( B ) MB49 tumor growth curves of irradiated tumors in the left hindlimb (left) and nonirradiated tumors in the right flank (right). ( C ) Survival curves of mice. The log-rank test is used to compare survival curves. ( D – G ) Proportions of CD45 + cells in the live cells ( D ), CD3 + cells in the CD45 + subpopulation ( E ), CD8 + CD4 − cells in the CD3 + subpopulation ( F ), and IFNγ + cells in the CD8 + CD4 − subpopulation ( G ) in single-cell suspensions of the irradiated and nonirradiated tumors on day seven in mice treated with CRT/postirradiation anti–PD-1 treatment compared to those of the nonirradiated tumors on day seven in mice treated with anti–PD-1 treatment alone ( n = 5/group). All data are shown as the mean ± standard error of the mean (SEM). Asterisks indicate p -values comparing two groups, as indicated in the figure. NS, not significant; * p < 0.05; ** p < 0.01; *** p < 0.001.

Article Snippet: MB49, a mouse bladder UC cell line, was purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Irradiation, Cell Analysis, Injection

Journal: International Journal of Molecular Sciences

Article Title: Combination of Cisplatin and Irradiation Induces Immunogenic Cell Death and Potentiates Postirradiation Anti–PD-1 Treatment Efficacy in Urothelial Carcinoma

doi: 10.3390/ijms22020535

Figure Lengend Snippet: The combination of cisplatin and irradiation increases HMGB1 protein secretion and cell surface expression of calreticulin protein in MB49 cells. ( A ) MB49 cells are treated with cisplatin at 0.6 mg/L and/or irradiated with a single fraction of 10 Gy. Medium is collected two and five days after treatment, and HMGB1 protein expression levels in each medium are examined in duplicate by ELISA ( n = 3/group). ( B ) MB49 cells are treated with cisplatin at 0.6 mg/L and/or irradiated with a single fraction of 10 Gy. Cells are collected, and cell surface calreticulin protein expression levels are examined by flow cytometry ( n = 3/group). ( C , D ) Seven days after inoculation MB49 cells in the left hindlimb, mice are treated with cisplatin at 3 mg/kg and/or irradiation with a single fraction of 10 Gy. Tumor sections before and after CRT ( n = 3/group) are stained for HMGB1 ( C ) and calreticulin ( D ). Left, representative images; right, comparison of the mean (± standard error of the mean [SEM]) number of HMGB1- or calreticulin-positive cells counted in five fields of view (FOV). All data are shown as the mean ± SEM. Asterisks indicate p values comparing two groups, as indicated in the figure. NS, not significant; * p < 0.05; ** p < 0.01.

Article Snippet: MB49, a mouse bladder UC cell line, was purchased from Merck KGaA (Darmstadt, Germany).

Techniques: Irradiation, Expressing, Enzyme-linked Immunosorbent Assay, Flow Cytometry, Staining, Comparison