Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: TFF3 overexpression enhances p53 transcriptional activity and protein expression and increases miR-34a expression with downstream reduction of EMP1 in Y79 RB cells. ( A ) Quantitative Real-time PCR confirmation of TFF3 lentiviral overexpression (Trefoil factor family peptide 3 (TFF3)) in Y79 cells compared to control cells (ctr). ( B ) Luciferase assays were performed with Y79 cells transiently transfected with TFF3 or empty vector control (ctr) in addition to wild-type PG13-Luc (wt PG13) or mutant MG15-Luc (mut MG15). Forced TFF3 expression leads to an increased luciferase signal upon p53 promotor activation in Y79 cells. ( C ) Western blot analysis showing increased p53 and TFF3 protein levels after TFF3 overexpression (TFF3). The indicated intensity ratios of p53 and TFF3 protein levels relative to β-actin levels were calculated using ImageJ software. ( D , E ) Quantitative real-time PCR analysis of miR-34a and EMP1 expression levels in Y79 cells compared to control cells after lentiviral TFF3 overexpression (ctr). Values are means of at least 3 independent experiments ± SEM. * p -value < 0.05, ** p -value < 0.01; statistical differences compared to the control group calculated by Student’s t -test or one-way ANOVA and Newman-Keuls post test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, Activity Assay, Expressing, Real-time Polymerase Chain Reaction, Luciferase, Transfection, Plasmid Preparation, Mutagenesis, Activation Assay, Western Blot, Software

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: Epithelial membrane protein 1 (EMP1) knockdown leads to reduced cell viability and proliferation and induces apoptosis in Y79 RB cells. ( A ) Western blot data confirmed decreased EMP1 protein levels after EMP1 knockdown (shEMP1) in Y79 cells. The CML cell line K562 served as an EMP1 positive control, ß-actin as a loading control. ( B , C ) Stably EMP1 knockdown Y79 RB cells (shEMP1) showed significantly decreased viability and proliferation levels compared to control cells (ctr) as revealed by ( B ) WST-1 assays and ( C ) BrdU stains. ( D ) EMP1 knockdown Y79 cells (shEMP1) displayed a significantly increased apoptosis rate compared to control cells (ctr) as revealed by DAPI stains. Values are means of 3 independent experiments ± SEM. *** p -value < 0.001 statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Western Blot, Positive Control, Stable Transfection

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: EMP1 overexpression leads to increased cell viability and proliferation and induces caspase-3/7 dependent apoptosis in Y79 RB cells. ( A ) Western blot data confirmed increased EMP1 protein levels after EMP1 overexpression (EMP1) in Y79 cells. The CML cell line K562 served as an EMP1 positive control, ß-actin as a loading control. ( B , C ) Stably EMP1 overexpressing Y79 RB cells (EMP1) showed significantly increased viability and proliferation levels compared to control cells (ctr) as revealed by ( B ) WST-1 assays and ( C ) BrdU stains. red: BrdU-labeled cells; blue: DAPI counterstaining ( D ) Growth curve analysis of EMP1 overexpressing Y79 RB cells showed a significant increase in cell growth rates. ( E ) EMP1 overexpressing Y79 cells (EMP1) displayed a significantly reduced apoptosis rate compared to control cells (ctr) as revealed by DAPI stains. ( F ) Caspase-3/7 activity was significantly reduced after EMP1 overexpression in Y79 RB cells (EMP1) compared to control cells (ctr). Values are means of at least 3 independent experiments ± SEM. ** p -value < 0.01; *** p -value < 0.001 statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, Western Blot, Positive Control, Stable Transfection, Labeling, Activity Assay

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: Effect of stable EMP1 overexpression on RB cell colony formation and anchorage independent growth. ( A ) Quantification of colony formation assays (CFA) showing a significant higher capacity of EMP1 overexpressing Y79 RB cells to form colonies (EMP1) compared to control cells (ctr). ( B ) Quantification of anchorage independent growth capacity of EMP1 overexpressing Y79 RB cells (EMP1) compared to control cells (ctr) as revealed by soft agarose assay. All photographs are taken 3 weeks after seeding EMP1 overexpressing and control Y79 RB cells. Values are means of at least 3 independent experiments ± SEM. *** p -value < 0.001; statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: Stable, lentiviral EMP1 overexpression increases tumor formation capacity of Y79 RB cells. ( A ) Photographs of CAM tumors in situ (left column), 3D tumor volume (middle column) and ruler measurements (in cm) of excised tumors (right column) revealing that tumors forming in the upper CAM after grafting EMP1 overexpressing Y79 RB cells were significantly larger compared to those developing from control cells (ctr). ( B , C ) Quantification of CAM assays by ( B ) tumor size, ( C ) tumor weight and ( D ) tumor volume. Values are means from at least 3 independent experiments (except for tumor volume which was measured exemplarily in one experimental setting) ± SEM. * p -value < 0.05, ** p -value < 0.01 statistical differences compared to the control group calculated by Student’s t -test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, In Situ

Journal: International Journal of Molecular Sciences

Article Title: p53, miR-34a and EMP1—Newly Identified Targets of TFF3 Signaling in Y79 Retinoblastoma Cells

doi: 10.3390/ijms20174129

Figure Lengend Snippet: MiR-34a and EMP1 overexpression leads to enhanced chemosensitivity. ( A ) Overexpression of miR-34a in Y79 RB cells leads to significantly increased apoptosis levels compared to control cells (ctr). Additional treatment with etoposide (Etop), cisplatin (CisP), or vincristine (Vin) significantly elevates the apoptosis levels compared to untreated miR-34a overexpressing Y79 RB cells. ( B ) Overexpression of EMP1 in Y79 RB cells leads to significantly decreased apoptosis levels compared to control cells (ctr). Additional treatment with etoposide (Etop) and vincristine (Vin) leads to higher apoptosis levels compared to untreated EMP1 overexpressing Y79 RB cells. Additional treatment with cisplatin (CisP) did not change the apoptosis level compared to untreated EMP1 expressing Y79 RB cells. Values are means of at least 3 independent experiments ± SEM. * p -value < 0.05, *** p -value < 0.001; statistical differences compared to the control group calculated by one-way ANOVA and Newman-Keuls post test.

Article Snippet: To generate a lentiviral EMP1 expression vector (pLenti CMV_ EMP1 ), the human EMP1 cDNA sequence was amplified from the RC208410 vector (Origene) using the forward primer 5′-C GAATTC TATGTTGGTATTGCTGGCTGG-3′ and the reverse primer 5′-CG CTCGAG TTATTTCTTTCTCAGGACCAG-3′ containing Eco RI and Xho I restriction sites (underlined).

Techniques: Over Expression, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Establishment and Validation of the Ascore Prognostic Signature. A Multivariate Cox coefficients for four ARGs (CERCAM, EMP1, GNLY, PTPRR) in the prognostic signature. B Ascore distribution among BLCA patients, sorted from lowest to highest. C Survival status categorized by Ascore for each BLCA patient. D Heatmap displaying expression levels of four genes in different Ascore groups. E Sankey diagram correlating clusters, Ascore groups, and BLCA survival status. F Kaplan–Meier analysis comparing overall survival between high and low Ascore groups in BLCA ( P < 0.0001). G Receiver Operating Characteristic (ROC) curves depicting Ascore signature’s predictive performance for 1, 3, and 5-year overall survival in BLCA, with the Area Under the Curve (AUC) values of 0.709, 0.724, and 0.745, respectively. (H–K) Kaplan–Meier analysis and time-dependent ROC curves in two external validation sets: GSE32548 and GSE32894

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: Biomarker Discovery, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Single-Cell RNA Sequencing Analysis of Ascore Distribution and Biological Significance in Bladder Cancer. A t-SNE plot showing seven main cell types distribution in the integrated dataset, with doublets manually annotated. B Dot plot of marker genes' expression levels in each cell type. C Ascore and four genes (CERCAM, EMP1, GNLY, PTPRR) expression and distribution across cell types. D t-SNE plot showing Ascore expression levels and patterns in each cell type. E Left plot: Six main epithelial cell subgroups visualized by t-SNE dimensionality reduction. Right plot: Ascore distribution and expression in epithelial cells, highlighting Subgroup0 and Subgroup2. F GO analysis of biological function differences between Subgroup0 and Subgroup2

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: RNA Sequencing, Marker, Expressing

Journal: Molecular Cancer

Article Title: Multi-cohort validation of Ascore: an anoikis-based prognostic signature for predicting disease progression and immunotherapy response in bladder cancer

doi: 10.1186/s12943-024-01945-9

Figure Lengend Snippet: Ascore Predictive Capability for Anti-PD-1 Immunotherapy Response in Gulou-Cohort2. A Representative immunohistochemistry (IHC) images illustrating the expression of four key genes (CERCAM, EMP1, GNLY, PTPRR) in two patients from Gulou-Cohort2 (Scale bars = 100 μm). Patient 4, who responded to anti-PD-1 therapy, had a low Ascore, in contrast to non-responder Patient 7, who had a high Ascore. B Distribution of Ascores among different response groups (CR: complete response; PR: partial response; SD: stable disease; PD: progressive disease; *** P < 0.001). C ROC curves comparing the predictive accuracy of Ascore (AUC = 0.913) versus PD-L1 expression in tumor-infiltrating immune cells (ICs) (AUC = 0.662). D Decision curve analysis (DCA) indicating the net benefit of using Ascore compared to evaluating ICs' PD-L1 expression. E Kaplan–Meier curves c showing a correlation between higher Ascore values in tissue samples and reduced survival rates ( P = 0.0194)

Article Snippet: Primary antibodies used in IHC are listed as follows: CERCAM (Proteintech®, 16,411–1-AP, 1:400); EMP1 (CUSABIO®, CSB-PA007648LA01HU, 1:400); GNLY (CUSABIO®, CSB-PA009627LA01HU, 1:400); PTPRR (Proteintech®, 17,937–1-AP, 1:100); PD-L1 (SP142 using the UltraPATH platform).

Techniques: Immunohistochemistry, Expressing