pdrgfp  (Addgene inc)

Journal: Journal of Translational Medicine

Article Title: TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer

doi: 10.1186/s12967-025-06648-3

Figure Lengend Snippet: TTI1 enhanced the resistance of RC to radiation. A Western blot and qRT-PCR were performed to analyse TTI1 protein and mRNA levels in many kinds of RC cells. B 72 h after irradiation, the MTT assay was used to detect the viability of DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids. C The clonogenic assay was used to detect proliferative ability of DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids after 0, 2, 6, 10 Gy irradiation. Representative images are shown in left panel. D HCT116 cells transfected with control vector or TTI1 expression plasmids were injected subcutaneously into the right dorsal flanks of nude mice. Xenograft tumours were treated with irradiation. Tumour size was measured every 3 days. E Tumours from euthanized mice were excised and weighed. F Western blot was performed to analyse TTI1 protein levels in those tumours. The error bars represent the mean ± SEM from three independent experiments. Data shown are mean ± SEM. * P < 0.05; ** P < 0.01; *** P < 0.001

Article Snippet: The DLD1 and HCT116 cell lines were transfected with pDRGFP(Addgene, Plasmid #26475) for the HR assay and with EJ5GFP(Addgene, Plasmid 44026) for the NHEJ assay.

Techniques: Western Blot, Quantitative RT-PCR, Irradiation, MTT Assay, Transfection, Control, Plasmid Preparation, Expressing, Clonogenic Assay, Injection

Journal: Journal of Translational Medicine

Article Title: TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer

doi: 10.1186/s12967-025-06648-3

Figure Lengend Snippet: TTI1 reduced radio-induced RC cells apoptosis. A Biological process enrichment was performed to analyse according to RNA-sequencing. B Flow cytometry was used to detect cell apoptosis rate of DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids after 6 Gy irradiation. After 48 h treatment, the cells were analysed by flow cytometry. Representative images are shown in left panel. The numbers of apoptotic cells (%, Q2 + Q4) were calculated and are presented in right panel. C Western blot analysis of apoptosis protein levels in DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids after 6 Gy irradiation. The error bars represent the mean ± SEM from three independent experiments. Data shown are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. IR irradiation

Article Snippet: The DLD1 and HCT116 cell lines were transfected with pDRGFP(Addgene, Plasmid #26475) for the HR assay and with EJ5GFP(Addgene, Plasmid 44026) for the NHEJ assay.

Techniques: RNA Sequencing, Flow Cytometry, Transfection, Control, Plasmid Preparation, Expressing, Irradiation, Western Blot

Journal: Journal of Translational Medicine

Article Title: TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer

doi: 10.1186/s12967-025-06648-3

Figure Lengend Snippet: TTI1 reduced DNA damage through HR repair pathway. A Western blot analysis of γH2AX protein level. The proteins were obtained at 0 min, 15 min, 30 min, 60 min, 120 min, 240 min and 480 min after 6 Gy irradiation exposure, respectively. B The comet assay was used to detect the extent of DNA damage in DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids at 12 h after 6 Gy irradiation exposure. Representative images are shown in lower panel. Quantifcation results are presented in upper panel. C Western blot analysis of HR repair and NHEJ pathway-related key proteins levels in DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids after 6 Gy irradiation. D Flow cytometry was used to detect the percentage of GFP-positive DLD1-DRGFP cells 48 h after 6 Gy irradiation exposure. The cells were transfected with shCtrl, shTTI1-1 or shTTI1-2 and then transduced with the pCBASceI plasmid 24 h later. E Flow cytometry was used to detect the percentage of GFP-positive HCT116-DRGFP cells 48 h after 6 Gy irradiation exposure. The cells were co-transfected with either control vector or TTI1 expression plasmids and pCBASceI plasmids. F Flow cytometry was used to detect the percentage of GFP-positive DLD1-EJ5GFP cells 48 h after 6 Gy irradiation exposure. The cells were transfected with shCtrl, shTTI1-1 or shTTI1-2 and then transduced with the pCBASceI plasmid 24 h later. G Flow cytometry was used to detect the percentage of GFP-positive HCT116- EJ5GFP cells 48 h after 6 Gy irradiation exposure. The cells were co-transfected with either control vector or TTI1 expression plasmids and pCBASceI plasmids. The error bars represent the mean ± SEM from three independent experiments. Data shown are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001. HR, homologous recombination; NHEJ, nonhomologous end joining; IR, irradiation

Article Snippet: The DLD1 and HCT116 cell lines were transfected with pDRGFP(Addgene, Plasmid #26475) for the HR assay and with EJ5GFP(Addgene, Plasmid 44026) for the NHEJ assay.

Techniques: Western Blot, Irradiation, Single Cell Gel Electrophoresis, Transfection, Control, Plasmid Preparation, Expressing, Flow Cytometry, Transduction, Homologous Recombination

Journal: Journal of Translational Medicine

Article Title: TTI1 contributes to radioresistance by activating ATM pathway in rectal cancer

doi: 10.1186/s12967-025-06648-3

Figure Lengend Snippet: TTI1 activated ATM signaling pathway. A ATM signaling pathway enriched in radioresistant tumour tissue. NES: Normalized Enrichment Score. B Western blot analysis of ATM signaling pathway-related key proteins levels in DLD1 cells transfected with shCtrl, shTTI1-1 or shTTI1-2 and HCT116 cells transfected with control vector or TTI1 expression plasmids after 6 Gy irradiation. C Co-immunoprecipitation was performed to analyse interaction between TTI1 and ATM. D Pull-down was performed to analyse direct interaction between TTI1 and ATM. E Co-immunoprecipitation was performed to analyse ATM monomer levels. HCT116 cells were infected with the ATM indicated expression plasmids and control vector or TTI1 expression plasmids and then treated by 0 Gy, 6 Gy irradiation. F Co-immunoprecipitation was performed to analyse ATM acetylation levels. HCT116 cells were infected with control vector or TTI1 expression plasmids and then treated by 0 Gy, 6 Gy irradiation. The error bars represent the mean ± SEM from three independent experiments. Data shown are mean ± SEM. *P < 0.05; **P < 0.01; ***P < 0.001, IR irradiation

Article Snippet: The DLD1 and HCT116 cell lines were transfected with pDRGFP(Addgene, Plasmid #26475) for the HR assay and with EJ5GFP(Addgene, Plasmid 44026) for the NHEJ assay.

Techniques: Western Blot, Transfection, Control, Plasmid Preparation, Expressing, Irradiation, Immunoprecipitation, Infection