recombinant human argonaute 2 eif2c2 protein  (Sino Biological)

Journal: Advanced Science

Article Title: Tryptophan As a New Member of RNA‐Induced Silencing Complexes Prevents Colon Cancer Liver Metastasis

doi: 10.1002/advs.202307937

Figure Lengend Snippet: Trp promotes miR‐193a‐mediated targeted mRNA cleavage by enhancing Ago2 RNase activity. A). SW620 cells were treated with or without biotinylated Trp (200 µM) for 12 h in Trp depletion medium. Biotin‐Trp was pulled down with streptavidin beads and Ago2 protein in the pulldown was analyzed by western blot. B). An in vitro Ago2 activity assay was performed using Ago2 and miR‐193a‐3p (500 nM) or miR‐193a‐3p mutant (mut, 500 nM) in the presence of Trp (200 µM), followed by adding 2.5 µg mRNA extracted from SW620 cells. Expression of caprin1 was determined by RT‐qPCR. P values were calculated by means of an ANOVA test. * p < 0.05. C). An in vitro Ago2 activity assay was performed using Ago2 wild‐type (WT) or mutant (mut) recombinant protein with miR‐193a‐3p in the presence of Trp (200 µM) or in Trp deletion medium, followed by adding 2.5 µg mRNA from SW620 cells. Expression of caprin1 was determined by RT‐qPCR. P values were calculated by means of an ANOVA test. * p < 0.05, NS, no significance. D). An in vitro Ago2 activity assay was performed using Ago2 and guide miR‐193a‐3p in the presence of Trp at the indicated dose, followed by adding mRNA from SW620 cells. Expression of caprin1 was determined by RT‐qPCR. P values were calculated by means of an ANOVA test. * p < 0.05, ** p <0.01, *** p < 0.001. E). An in vitro Ago2 activity assay was performed using Ago2 and guide miR‐193a‐3p or Trp‐miR‐193a‐3p, followed by adding mRNA from SW620 cells. The reaction was stopped at the indicated time point. Expression of caprin1 was determined by RT‐qPCR. P values were calculated by means of an ANOVA test. * p < 0.05, *** p < 0.001. F). SW620 were transfected with Flag‐Ago2‐WT or Flag‐Ago2 mutant (P590G and R688S) plasmid for 48 h. Transfected SW620 cells were cultured in Trp depleted medium for 12 h, and then treated with Trp (200 µM) or PBS as a control for culturing additional 12 h before the cells were harvested. Ago2 complex was pulled down using protein G beads cross‐linked with anti‐Flag M2 antibody (Sigma–Aldrich) and eluted by 3xFlag peptide (Rockland Immunochemicals, PA). An Ago2 activity assay was performed using Ago2 complex as mentioned and guide miR‐193a‐3p in the presence of Trp, followed by adding mRNA from SW620 cells. The reaction was stopped at the indicated time point. Expression of caprin1 was determined by RT‐qPCR. P values were calculated by means of an ANOVA test. * p < 0.05, NS, no significance. G). Trp concentration in the adjacent normal tissue or in the tumor tissue of patients in cancer stage I/II (circles) and stage III/IV (squares) were determined by HPLC. * p < 0.05, ** p < 0.01. H). MiR‐193a‐3p level in the adjacent normal tissue or in the tumor tissue of patients in cancer stage I/II (circles) and stage III/IV (squares) were determined by RT‐qPCR. * p < 0.05, ** p < 0.01. I,J). Fluorescence in situ hybridization of tumor sections from human colon samples stained with biotinylated Ago2 (green) were overlayed with Trp (red) and miR‐193a‐3p (purple). Five random fields were photographed in z‐stack and representative 3D reconstruction images were obtained using “surface” plug‐in in Imaris software. Scale bars, 40 µm. K) The percentage of co‐localization intensity of Ago2, Trp and miR‐193a‐3p were analyzed by Image J software and the results is shown. L). Cartoon figure shows the structure of the Ago2 protein containing a tandem tryptophan pocket bound to Trp‐miR‐193a‐3p complex.

Article Snippet: [ , ] Murine Ago2 recombinant protein was purchased from SinoBiological Inc. (Cat#: 50683‐M07B) and human Ago2 recombinant protein was provided as a gift from Dr. Ian J. MacRae (The Scripps Research Institute, CA, USA).

Techniques: Activity Assay, Western Blot, In Vitro, Mutagenesis, Expressing, Quantitative RT-PCR, Recombinant, Transfection, Plasmid Preparation, Cell Culture, Control, Concentration Assay, Fluorescence, In Situ Hybridization, Staining, Software