LIN-65, but not ARLE-14, is required for MET-2 localization, focus formation, and stability. (A) Confocal images of MET-2::FLAG::mCherry-expressing embryos after RNAi for lin-65, arle-14, or empty vector (vector). Bar = 5 µm. (B) Quantitation of MET-2::FLAG::mCherry foci in embryos as shown in A using automated image analysis. N = 3, n (vector) = 873, n (lin-65) = 1,294, and n (arle-14) = 1,209; P values are indicated above scatter plots and were calculated using a Kruskal–Wallis test with Dunn’s correction for multiple testing. (C) Quantitation of MET-2::FLAG::mCherry foci distance to nuclear rim as shown in A using the three-zone method (see Fig. 2 and Meister et al. [2010a]). N = 3, n (vector) = 4,822, and n (arle-14) = 7,397. (D) Images of MET-2::FLAG::mCherry in wild-type and lin-65(gw1465) mutant embryos with nuclear rim visualized by LEM-2::GFP. Bar = 5 µm. (E) Quantitation of MET-2::FLAG::mCherry fluorescence intensity in the cytoplasm and the nucleus in wild-type and lin-65(gw1465) embryos (n = 118, P values were calculated using two-sided Wilcoxon signed-rank test and indicated above violin plots). (F) Example Western blot (top) and quantification (bottom) of MET-2::FLAG::mCherry protein levels in wild-type, lin-65(gw1465), and lin-65(n3441) embryos from Western blots (N = 4) probed as indicated and normalized to endogenous MRG-1 abundance in wild-type embryos. P values in met-2::FLAG::mCherry(gw1419) versus wild-type: lin-65(gw1465) = 0.042, lin-65(n3441) = 0.036; bars indicate mean signal. (G) As in F, except that H3K9me2 levels are quantified, in wild-type, met-2(n4256), lin-65(gw1465), lin-65;met-2, and lin-65(n3441) embryos. N = 3, P values versus wild-type: met-2 = 0.015, lin-65(gw1465) = 0.028, lin-65;met-2 = 0.018, lin-65(n3441) = 0.007; bars indicate mean signal. (H) Number of viable progeny of strains indicated in G cultured at 20°C (N = 2, n = 50; P values were calculated using two-sided ANOVA and indicated above boxplots).