Supplemental Figure 4 (Related to Figure 4)A) Single molecule(sm) mRNA FISH analysis of gfp mRNA in miR-228 reporter strains.Images are maximum intensity projections of z-stacks. DAPI (blue); GFP::H2B (green);smFISH against gfp sensor mRNA (magenta). Dashed lines delineate the gonadboundary. Scale bars are 20um. B) Quantification of the number of foci observedfollowing smFISH analyses of gfp mRNA in the control and sensor reporters. The resultis presented as the mean calculated from 10 different worms for each condition anderror bars represent standard deviation from the mean. The number of foci wascompared between mutated and sensor reporters. ***P-value<0.0001. C) Analysis ofPGL-1::RFP and gfp mRNA localization upon knock-down of P granule factors.Fluorescence microscopy and single molecule mRNA FISH analysis of gfp mRNA inmutated miR-228 reporter germlines upon RNAi against indicated genes. DAPI (blue),FISH against gfp mRNA (magenta) and merge channels (right). Scale bars are 5μm. D)Single molecule mRNA FISH analyses of gfp mRNA localization in the pachytene regionof distal gonad arms. The proportion of gfp mRNA foci localized inside the nucleus (in),on the nuclear periphery (per) or outside the nucleus (out) were quantified for at least340 nuclei/experimental condition. The error bars represent the 95% confidence intervalfrom three independent experiments. E-F) Left: Quantification of
glh-1,
pgl-1 and deps1 mRNA expression upon their respective RNAi knock-down, compared to control RNAi.mRNA levels were measured by quantitative RT-PCR of mutated (E) and sensor (F)miR-228 reporters. Young adult stage whole worm RNA extracts were used, and mRNAlevels were normalized to
tba-1 mRNA level. The error bars represent the 95%confidence interval from three independent experiments. *P-value<0.05. Right:Quantification of
glh-1 mRNA expression upon
pgl-1 and
deps-1 RNAi knock-down,compared to control RNAi. mRNA levels were measured by quantitative RT-PCR ofmutated (E) and sensor (F) miR-228 reporters. Young adult stage whole worm RNAextracts were used, and mRNA levels were normalized to
tba-1 mRNA level. The errorbars represent the 95% confidence interval from three independent experiments. G)Fluorescence microscopy of miR-228 mutated or sensor reporter germlines upon RNAiagainst indicated genes. Dashed lines delineate the gonad boundary. Scale bars are 20μm.