Fig. 2: Germline AGO2 mutations impair shRNA-mediated silencing.

a–c AGO2-deficient HEK293T cells were transfected with mRFP-Shank3 (a), mRFP-DDX1 (b), or GFP-tagged δ-catenin (c) along with shRNA constructs targeting Shank3 (a; in pLVTHM vector coexpressing GFP), DDX1 (b; in pSuper) or δ-catenin (c; also in pSuper), and Flag/HA-tagged (Shank3, δ-catenin) or GFP-tagged (for DDX1) AGO2 variants as indicated. Efficient transfection (>70 % of cells) was verified by fluorescence microscopy using appropriate fluorophores (GFP in (a) and (b); RFP in (c). Cells were lysed and lysates were analysed by Western Blotting using the antibodies indicated. In each case, each experiment was repeated eight times with similar results. d Quantification of the representative immunoblots shown in (a–c). The expression levels are relative to those in control cells without an AGO2 construct (first lane in each immunoblot). e Quantification of a second set of AGO2 mutants. For (d, e), data are means + SEM. Data for mutant and control conditions were compared to wt. *, **, ***p < 0.05, 0.01, 0.001, respectively; n = 8 biologically independent experiments in (d); one-way-ANOVA, followed by Holm-Sidak’s multiple comparisons test; n = 6–14 biologically independent experiments in e for Shank3; mixed-effects analysis, followed by Holm-Sidak’ multiple comparisons test; n = 6 biologically independent experiments in (e) for δ-catenin; one-way ANOVA, followed by Dunnett’s multiple comparisons test. f Knockdown of DDX1 with DDX1 shRNA was performed as in (b), but using F/H-tagged AGO2 in combination with GFP, or in combination with GFP-tagged AGO2 variants. Note that the GFP-tagged AGO2 mutants do not interfere with the knockdown capacity of F/H-AGO2 WT. n = 8 biologically independent experiments; data are means +/− SD; ****p < 0.0001; one-way ANOVA, followed by Dunnett’s multiple comparisons test. Source data are provided as a Source Data file.