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. 2017 Jul 5;20(1):173–187. doi: 10.1016/j.celrep.2017.06.027

Figure 5.

Figure 5

LIMD1 Is Required for Phospho-S387-AGO2 to Engage TNRC6A/DDX6 for miRNA-Mediated Silencing

(A and B) (A) psiCHECK-2-miR99/100 reporter assay in HeLa CRISPR-Cas9 LIMD1+/+ or (B) LIMD1−/− cell lines transfected with non-targeting (SCR) or AGO2 siRNA and EYFP-AGO2, EYFP-AGO2S387A, or EYFP-AGO2S387E.

(C) miR99/100 reporter assay in in above HeLa CRISPR-Cas9 cell lines transfected with non-targeting (SCR) or AKT3 siRNA.

(D) PLA analysis of endogenous phospho-AGO2 (S387):TNRC6A interaction in situ in HeLa CRISPR-Cas9 LIMD1+/+ or LIMD1−/− lines. PLA signal orange, cells stained with DAPI (top); PLA signal white for visual clarity (bottom). Scale bars, 10 μm.

(E) Quantification of PLA interaction events in (D), displayed as stacked histograms. Data are mean ± SEM, n = 3, total of 200 cells determined using the chi-square test.

(F) PLA analysis of phospho-AGO2 (S387):DDX6 interaction in above HeLa CRISPR-Cas9 cell lines.

(G) Quantification of (F) as in (E).

Unless otherwise stated, data are mean ± SEM, n = 3, ∗p < 0.05, ∗∗p < 0.001, ∗∗∗p < 0.0001 according to the Student’s t test.