Fig. 3: Single-molecule imaging of RISC-binding.
From: Single-molecule imaging of microRNA-mediated gene silencing in cells
a Schematic of the IF-FISH experiment to visualize RISC-binding at single-mRNA resolution. Cyan and magenta spots represent RISC and reporter mRNAs, respectively. b The images of U2OS cells expressing the 8Ă— miR-21 mutant reporter (left) and the 8Ă— miR-21 reporter (right). Reporter mRNAs (magenta, top) and AGO (cyan, middle) were labeled by IF-FISH. Merged images are shown at the bottom. White and black arrowheads indicate RISC-positive and RISC-negative mRNAs, respectively. Scale bar, 1 μm. c, d RISC-binding mediated by miR-21. Images were analyzed using CellProfiler and FISH-quant. Then, RISC-binding efficiency was calculated by division of the total AGO intensity on mRNAs by the number of mRNAs as described in Supplementary Fig. 7 (see also Methods). The results of bulk analysis (c) and single-cell analysis (d) are shown. In d, each circle represents a single cell (n = 50 for each condition), while red lines represent the medians. The p-value of one-tailed Mann–Whitney test is shown. e Increase of the fraction of RISC-positive mRNAs by miR-21. The fraction of RISC-positive mRNAs was calculated as described in Supplementary Fig. 7 (see also Methods). Each circle represents a single cell (n = 50 for each condition), while red lines represent the medians. The p-value of one-tailed Mann–Whitney test is shown. f The ratio of RISC-negative and RISC-positive mRNAs. All mRNAs were classified into RISC-negative or RISC-positive mRNAs based on 3D-colocalization analysis. g The histogram of AGO intensity. The intensities of free AGO spots (dashed lines) and of AGO spots on mRNAs (solid lines) are shown. The p-values of Dunn’s multiple comparisons test are shown. n.s., not significant. Source data are provided as a Source Data file.
