Extended Data Fig. 4: PEI granules specifically recruit WAGO-3.
a,b, Confocal maximum intensity projections of an adult hermaphrodite (a) and gastrula-staged embryo (b) expressing indicated proteins. Zooms show perinuclear co-localization of GFP::3xFLAG::WAGO-3 and PGL-1::mTagRFP-T in meiotic (a) and primordial (b) germ cells. Z2/Z3 are the primordial germ cells of C. elegans. Scale bars: 20 μm (a, adult), 20 μm (b, embryo), 10 μm (a, zoom), 4 μm (b, zoom). c, Quantification of GFP::3xFLAG::WAGO-3 foci number within indicated, male-derived germ cells (n = 10 cells pooled from two independent experiments, for each condition). Statistically significant differences were determined by one-way ANOVA (p ≤ 0.001) followed by Tukey’s honestly significant difference post hoc test (p ≤ 0.05). Different letters represent significant differences. Note that the values for primary spermatocytes and budding spermatids (c) are the same as those shown in Fig. 3j (FL) and Extended Data Fig. 10f (wild-type), and Fig. 4n (FL) and Fig. 7j (wild-type), respectively. Secondary spermatocyte and budding spermatid stages are separated into ‘c’ and ‘s’. c: coupled; due to incomplete cytokinesis, leaving the two sister cells coupled and both cells were analyzed; s: separate, each of the coupled cells in ‘c’ was analyzed individually. Boxplot centre and box edges indicate median and 25th or 75th percentiles, respectively, while whiskers indicate the median ± 1.5 x interquartile range. d, Co-immunoprecipitation experiments using whole-worm extracts of late-L4 stage hermaphrodites analyzed by Western blotting. Sample processing control was run on a different gel. Data represent two biologically independent experiments. e-j, Confocal micrographs of an adult male (f) and late-L4 stage hermaphrodites (e,g-j) expressing indicated proteins. sc – spermatocyte, st – spermatid, rb – residual body. Scale bars: 10 μm (e-j). a,b,e-j, Images represent two biologically independent experiments. k-n, Co-localization analyses between PEI-1::mTagRFP-T and DEPS-1::GFP (k), GFP::3xFLAG::WAGO-1 (l), GFP::3xFLAG::CSR-1 (m) and GFP::3xFLAG::ALG-3 (n) based on the images shown in g-j, respectively (n = 10 worms for each condition). X and Y axes indicate fluorescence intensity. PC: Pearson’s correlation coefficient. Exact P values (c), unprocessed original scans of blots and the source data for all graphical representations are provided.
