Fig 2: PLA for the validation of new p65 interactors: Analysis of endogenous NF-κB p65/DCP1a complexes. (A) Double-immunofluorescence analysis of DCP1 and p65 in HeLa cells stably transfected with pSUPER.puro. (B–D) PLAs were used to determine the interaction of endogenous DCP1a with p65 NF-κB in HeLa cells before and after stimulation with IL-1α (10 ng/mL) at different times. The specificity of the obtained PLA signals was validated by omitting one or both primary antibodies. (E–G) Additionally, specificity of PLA was assessed in cells transiently transfected for 24 h with a p65 shRNA construct or with empty vector (pSUPER.puro) control. Representative images, the summary of PLA quantification and statistical tests are shown. (E–G) Data were pooled from three independent experiments (50 cells each). Distribution of PLA signals and the summary and statistics of all relevant data are depicted graphically and as tables as before. (H) The p65 knockdown was also confirmed in cell extracts by immunoblotting.

Fig 3: DCP1a is required for efficient nuclear translocation of p65 NF-?B. (A) HeLa cell lines with CRISPR/Cas9-mediated suppression of DCP1a (?DCP1a) or cells stably transfected with the control vector (px459) were incubated for the indicated times with IL-1a (10 ng/mL) or were left untreated. Cytosolic and nuclear extracts were prepared and the presence of p65 and DCP1a proteins was examined by immunoblotting. Anti-tubulin, ß-actin, and P(S2)-RNA polymerase II antibodies were used to control for purity of the fractions and for equal loading. Shown is one out of two independent experiments with compatible results. (B) Shows relative quantification of mean p65 protein amounts +/- SD from two independent experiments. (C) The same cells were seeded at two different densities and cell numbers were assessed at the indicated time points. The graphs show cell numbers and corresponding fold changes.

Fig 4: Antagonistic interaction between NANOS2 and DAZL in XY germ cells.(a) Venn diagrams show the overlap among DAZL-associated gene probes in XX (magenta) and XY gonads (green), and NANOS2-associated probes in XY gonads (blue) at E14.5. Probes showing fourfold higher levels (P<0.05) in the IP than in the input were selected. (b) RIP and RT-qPCR analysis of selected DAZL-associated genes. RIP was performed with an anti-DAZL antibody. The y axis represents the relative enrichment of IP to input. Error bars, ±s.d. (n=3). (c) RIP performed with anti-DAZL antibody using Nanos2+/+and Nanos2−/− XY gonads at E14.5. The relative enrichment of selected DAZL-associated mRNAs in IPs was measured by qPCR. To compare the amounts of the precipitated mRNAs in the Nanos2+/+ and Nanos2−/− gonads, sample volumes for cDNA synthesis were normalized to the immunoprecipitated DAZL protein. Error bars, ±s.d. (n=3). Significance levels of changes are indicated (Mann–Whitney U-test; **P<0.005, *P<0.05). (d) Correlation analysis between the association of DAZL and the expression changes of the DAZL and NANOS2-associated genes in Nanos2−/− XY gonads. The x axis represents the difference in IP/input of DAZL in XX versus XY gonads. The y axis represents relative expression changes of the genes in Nanos2−/− versus Nanos2+/+ gonads. Red dots indicate 274 overlapping probes in a showing statistical reliability (two-tailed Student's t-test; P<0.05) in our previous microarray analyses21. r, correlation efficient. (e) RNA-immunoprecipitation and RT-qPCR analysis, using anti-NANOS2 antibody, of XY gonads at E15.5 as same with Fig. 1g. (f) Immunostaining of XY germ cells at E15.5. CDH1 was used as a germ cell marker. Arrowheads indicate granules where NANOS2 and DCP1a are co-localized. Scale bars, 5 μm. (g,h) DCP1a (g) and NANOS2 (h) granules in XY germ cells quantified using single planes of confocal photomicrographs. Horizontal bars indicate the mean values. (i) The proportion of DCP1a granules co-localized with NANOS2 granules. (g–i) The significance of changes is indicated (Steel–Dwass all-pairs non-parametric test; ***P<0.0005). IP, immunoprecipitate.

Fig 5: UBAP2L is localized to SGs.a HeLa cells treated with 0.5 mM arsenite (AS) for 30 min or 0.3 M sorbitol for 30 min were immunostained for UBAP2L together with G3BP1, TIAR, eIF4E, or PABPC1 (scale bar = 10 µm). b HeLa cells were transfected with plasmids encoding GFP or GFP-UBAP2L, and 24 h later, the cells were treated with or without 0.5 mM arsenite (AS) for 30 min and immunostained for GFP and G3BP1(scale bar = 10 µm). c HeLa cells were treated with 0.5 mM arsenite for 30 min in the presence or absence of cycloheximide (CHX) and immunostained for G3BP1 and UBAP2L (scale bar = 10 µm). d Cells treated with 0.5 mM arsenite were immunostained for DCP1A and UBAP2L (scale bar = 10 µm). e Fluorescence photo bleaching recovery of a GFP-UBAP2L-expressing SG was monitored by confocal microscopy in the presence of 0.5 mM arsenite. The arrow shows the monitored SG. The time after irradiation is shown in each photograph (scale bar = 10 µm).