Extended Data Fig. 10: SMO-1 suppresses piRNA expression and increases UAD-2 mobility.
From: piRNA gene density and SUMOylation organize piRNA transcriptional condensate formation
a-c, Subcellular localization of UAD-2::GFP, TOFU-4::GFP and TOFU-5::GFP with mCherry::H2B in pachytene cells of WT worms fed L4440 and smo-1 dsRNA at 20 °C. All images were taken by the Leica THUNDER imaging System and deconvoluted using Leica Application Suite X software (version 3.7.4.23463). All images are representative of more than three animals (see also in Extended Data Fig. 10m). d-f, Relative fluorescence unit intensity (RFU) indicated by dashed lines along chromosome IV of (a-c). Source data are provided as a Source Data file. g, Expression levels of total piRNAs from LG IV in specified adult animals grown at 20 °C, normalized as reads per million+1. h, Boxplots illustrating log2(piRNA reads per million+1) for the indicated adult animals from one biological replicate. The box itself represented the interquartile range between 5% and 95%, thereby encompassing the middle 90% of the data set. The central horizontal line within the box represents the mean value. Error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Outliers are individually marked as points, representing data points that fall outside the range of mean±1.5 SD (see also in Extended Data Fig. 10j). Statistical significance was determined using a paired-sample t test. i, qRT-PCR analysis of tofu-3 mRNA in wild-type animals and mCherry::TOFU-3 animals. The levels were normalized to eft-3. Quantification of qRT-PCR data from n = 3 independent animals, error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Statistical significance was determined using a paired-sample t test. Source data are provided as a Source Data file. j. Boxplots illustrating log2(pre-piRNA reads per million+1) for wild-type worms and mCherry::TOFU-3 animals at 25 °C from one biological replicate. Statistical significance was determined using a paired-sample t test. k, Fluorescence recovery after photobleaching (FRAP) of UAD-2::GFP under L4440 and smo-1 RNAi was conducted using a Zeiss LSM980 confocal microscope. All images are representative of 6 animals. l, Graphs presenting the relative fluorescence unit intensity (RFU) of the control area and bleached area of UAD-2 under L4440 and smo-1 RNAi. Quantification of FRAP data from n = 6 independent animals, error bars represent the mean ± 1.5 standard deviations (SD), indicating the variability of the data around the mean. Source data are provided as a Source Data file. m, Images showing germline nuclei expressing UAD-2::GFP. After release by needle disruption, the germline was imaged within 5 minutes using a Leica Thunder imaging system. Germlines were treated with 10% 1,6-hexanediol to prohibit phase separation.
