tnt quick coupled transcription/translation system kit l1171  (Promega)

Journal: Journal of Advanced Research

Article Title: Targeting ATM enhances radiation sensitivity of colorectal cancer by potentiating radiation-induced cell death and antitumor immunity

doi: 10.1016/j.jare.2024.12.023

Figure Lengend Snippet: Targeting ATM promotes mitochondrial damage post-irradiation to facilitate STING pathway activation. A. The cytosolic abundance of the MTATP8 DNA sequence was measured by qPCR 24 h post-IR. B. Mitochondrial morphological changes were examined using transmission electron microscopy in SW620 cells collected 24 h post-IR. C. Flow cytometric analysis detected changes in mitochondrial membrane potential, with a reduced green/red fluorescence intensity ratio (FITC/PE) indicating mitochondrial depolarization. Samples were collected 24 h after treatment with IR (6 Gy), AZD0156 (2 uM), or their combination. D. Mitochondrial ROS levels were examined by flow cytometric detection 24 h post-IR. E. Control and rho 0 cells (mtDNA-depleted) were treated with IR (6 Gy) alone or combined with AZD0156 (2 uM). Cells were co-stained with MitoTracker Red CMXRos (mitochondria, red), anti-TFAM (green), and DAPI. F. Western blot analysis of STING signaling protein expression in SW480 and SW620 control and rho 0 cells treated with IR alone or in combination with AZD0156. Total and phosphorylated levels of STING, TBK1, and IRF3 were evaluated 48 h post-treatment. G. qRT-PCR analysis of IFNB1, CXCL10, and CXCL11 expression in control and rho 0 cells treated with IR alone (6 Gy) or in combination with AZD0156 (2 uM), 24 h post-IR. H. ELISA analysis of IFNB1, CXCL10, and CXCL11 levels in cell supernatants from control and rho 0 cells treated with radiation alone or in combination with AZD0156. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The ELISA kits used included the Mouse Aspartate Aminotransferase (AST) ELISA Kit, Mouse Alanine Aminotransferase (ALT/GPT) ELISA Kit (CUSABIO, China), Mouse Troponin T (TnT) ELISA Kit (MEIMIAN, MM-44145H1), and Mouse Creatine Kinase Isoenzyme MB (CK-MB) ELISA Kit (MEIMIAN, MM-43703H1).

Techniques: Irradiation, Activation Assay, Sequencing, Transmission Assay, Electron Microscopy, Membrane, Fluorescence, Control, Staining, Western Blot, Expressing, Quantitative RT-PCR, Enzyme-linked Immunosorbent Assay

Journal: Journal of Advanced Research

Article Title: Targeting ATM enhances radiation sensitivity of colorectal cancer by potentiating radiation-induced cell death and antitumor immunity

doi: 10.1016/j.jare.2024.12.023

Figure Lengend Snippet: Reducing ROS accumulation after radiation attenuates immune activation induced by ATM inhibition. A. Flow cytometry analysis of ROS generation in DCFH-DA-stained SW480 and SW620 cells treated with IR (6 Gy), AZD0156 (2uM), or their combination. B. Fluorescent imaging of SW480 and SW620 cells demonstrating the effect of AZD0156 on intracellular ROS production following IR (6 Gy). ROS is shown as green fluorescence. C. The ROS scavenger NAC (5 mM) was used to reduce IR-induced or IR plus AZD-induced ROS levels in SW480 cells. D. The abundance of the cytoplasmic MTATP8 DNA sequence after NAC treatment was analyzed by qRT-PCR. E. Cells were treated with IR alone or IR combined with AZD0156, with or without the addition of NAC. Expression levels of IFNB1, CXCL10 and CXCL11 were analyzed by qRT-PCR 24 h post-IR. F. ELISA quantified CXCL10, CXCL11 and IFNB1 levels in cell supernatants from the four treatment groups. G. Western blot analysis was used to assess protein expression in the ATM/CHK2/Beclin signaling pathway in SW480 and SW620 cells treated with control, IR (6 Gy), AZD0156 (4uM), or their combination.Total and phosphorylated levels of ATM, CHK2, Beclin1, and autophagy-related proteins p62 and LC3 were assessed by western blot after 48 h. H. Schematic diagram of ex vivo chemotaxis assay of CD8+ T cell activation and migration. I. Flow cytometry was used to quantify the number of CD8+ T cells that migrated to the lower chamber. The supernatants were derived from tumor cells treated with Non-IR, IR alone, IR combined with NAC, IR combined with AZD0156, or a combination of all three treatments. Data are represented as mean ± SEM. *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001. (For interpretation of the references to colour in this figure legend, the reader is referred to the web version of this article.)

Article Snippet: The ELISA kits used included the Mouse Aspartate Aminotransferase (AST) ELISA Kit, Mouse Alanine Aminotransferase (ALT/GPT) ELISA Kit (CUSABIO, China), Mouse Troponin T (TnT) ELISA Kit (MEIMIAN, MM-44145H1), and Mouse Creatine Kinase Isoenzyme MB (CK-MB) ELISA Kit (MEIMIAN, MM-43703H1).

Techniques: Activation Assay, Inhibition, Flow Cytometry, Staining, Imaging, Fluorescence, Sequencing, Quantitative RT-PCR, Expressing, Enzyme-linked Immunosorbent Assay, Western Blot, Control, Ex Vivo, Chemotaxis Assay, Migration, Derivative Assay