Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: Impact of the modulation of TYRO3 expression on the radiosensitivity of bladder cancer cell lines. ( a – c ) Representative clonogenic survival curves of RT112 and 5637 cell lines ( upper panel). 48 h after transfection with siLUC (control, red), siTYRO3#4 (blue), siTYRO3#801 (green) or diluted concentration of siRNA#1 (black) cells were exposed to increased doses of gamma-rays. In each case, downregulation was confirmed by western blot at 48 h after transfection. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( d ) Representative survival curves of UM-UC-3 control (empty plasmid, red) and TYRO3 over-expressed cell lines (TYRO3 encoding- plasmid, purple) ( upper panel). The upregulation was confirmed by western blot. The corresponding D 10 values were calculated after fitting the experimental data to the classical linear-quadratic equation ( lower panel); ( e ) Phosphorylated TYRO3 shown in western blot ( lower panel) after immunoprecipitation of the cell extracts with phospho-Tyrosine antibodies and its relative expression in 5637 cells quantified using Image-J software and normalized to that of total TYRO3 ( upper panel). Data represents mean ± SD of 3 independent experiments. Unpaired t-test analysis: * p < 0.05; ** p < 0.005, ns: not significant.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Expressing, Transfection, Control, Concentration Assay, Western Blot, Plasmid Preparation, Immunoprecipitation, Software

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on Ionizing Radiation-Induced Foci and DNA damage. γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 downregulated RT112 ( a ) or 5637 ( c ) cells (scale bar 5 microns); Quantification of cells containing more than 10 γH2AX foci at 24 h after 2 Gy of irradiation in the downregulated RT112 ( b ) and 5637 ( d ) cell lines; ( e ) γH2AX foci visualized after 24 h of 2 Gy irradiation in TYRO3 overexpressing UM-UC-3 cell line (Scale bar 5 microns); ( f ) Quantification of cells containing more than 10 γH2AX foci at 30 min and 24 h after 2 Gy of irradiation in TYRO3 overexpressing cell lines. The data shown above is from three different experiments and error bars represent the SD. Unpaired t -test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005; **** p < 0.0005, ns: non-significant. Representative images of the alkaline Comet assay performed on TYRO3 downregulated RT112 ( g ) and 5637 ( h ) cells and the resulting Olive tail moments analysis in TYRO3 downregulated RT112 ( i ) and 5637 ( j ) cells irradiated at 6 Gy. The data shown here is from three independent experiments analyzing 200 nucleus per condition per experiment, horizontal bars represent the median values. Kruskal-Wallis nonparametric tests with Multiple comparisons were used: * p < 0.05; ** p < 0.005, ns: non-significant; ( k ) western blot of DNA damage repair proteins; TYRO3 was downregulated, irradiated at 6 Gy and the lysates were prepared and analyzed 0.5–24 h after.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Alkaline Single Cell Gel Electrophoresis, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 modulation and its impact on DNA damage response pathways. ( a ) Volcano plot showing the fold change (Log2 Ratio) versus negative log of the p-value of differentially expressed genes after Nanostring analysis between BCa (RT112 and 5637) 6 Gy irradiated cells versus BCa 6 Gy irradiated cells after complete TYRO3 knock-down (siTYRO3#4 and siTYRO3#801). Significant: p -value < 0.05 ( b ) Log2 ratio of the significantly expressed genes ( p < 0.05) after Nanostring analysis between the two groups. The name of the up- or downregulated genes are listed on the left. On the right are the corresponding Nanostring gene annotations. ( c ) Protein-protein association network of the up and downregulated genes assessed using the STRING database.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Knockdown

Journal: International Journal of Molecular Sciences

Article Title: Tyro3 Targeting as a Radiosensitizing Strategy in Bladder Cancer through Cell Cycle Dysregulation

doi: 10.3390/ijms23158671

Figure Lengend Snippet: TYRO3 downregulation affects cell cycle following irradiation. Analysis of the cell cycle distribution 24 h after 6 Gy irradiation on TYRO3-downregulated RT112 ( a ) and 5637 ( b ) cells. Comparison of percentage (%) of cells in G2/M in RT112 ( c ) and 5637 ( d ) cells. Data shown is from three different experiments and error bars represent the SD. Unpaired t-test analysis: * p < 0.05; ** p < 0.005; *** p < 0.0005, ns: non-significant. ( e ) western blot of cell cycle proteins 30 min and up to 24 h after irradiation.

Article Snippet: The human BCa-derived cell lines 5637, RT112, UM-UC-5, UM-UC-9, VM-CUB-1, were obtained from DSMZ (Heidelberg, Germany), and UM-UC-3 from ATCC.

Techniques: Irradiation, Comparison, Western Blot