Fig 2: Iws1 is required for the expression of Nanog. (A) EU-derived fluorescence was used to assess the global transcription in embryos electroporated with the indicated siRNAs. The fluorescence intensity of EU is highest in 2-cell stage siControl embryos and exhibits drastic decreases in siIws1 and siSupt6 embryos at 96 hphCG. At least 20 embryos were used for each experiment. (B) Immunofluorescent detection of Pou5f1 (Oct4), Cdx2 and Nanog in siControl, siIws1 and siSupt6 embryos. Representative images of E3.5 embryos are shown. DNA was counterstained with DAPI. At least 30 embryos were used for each experiment. (C) RT-qPCR analysis of the Pou5f1, Cdx2 and Nanog mRNA in siControl, siIws1 and siSupt6 embryos at the indicated times after hCG injection. The mRNA expression of H2afz was used as an internal control. The mRNA expression levels of Pou5f1, Cdx2 and Nanog in siControl embryos at 44 hr, 70 hr, and 44 hr, respectively, were defined as 1. One-hundred embryos were used for each analysis. (D) PLA showing the apparent interaction of Prmt5 and Iws1 in the cell nuclei of representative 4-cell stage embryos. (E) PLA showing that Prmt5 and Nanog interact in early embryos and that Iws1 is required for this interaction. DNA was counterstained with DAPI. At least 20 embryos were used for each experiment. Scale bars: 20 µm.

Fig 3: LEDGF/p75 and its IBD-interacting partners are differentially upregulated in DTX-resistant PCa cell lines. Upper panels: immunoblots showing upregulation of (A) LEDGF/p75, (B) JPO2, (C) c-MYC, (D) menin, (E) MLL, (F) IWS1, (G) ASK1, (H) PogZ, (I) Med-1, and (J) HRP2 in DTX-resistant PC3-DR and DU145-DR cells compared to their parental, drug-sensitive counterparts. Bottom panels: bar graphs showing quantification of fold change in protein expression from at least three independent experiments per cell line via densitometric ImageJ analysis, with values normalized to ß-actin. Paired t-test statistical analysis revealed that protein expression in DTX-resistant PCa cells achieved statistical significance compared to controls (* p < 0.05, ** p < 0.01). Due to the very low expression of some of the proteins in the parental PC3 and DU145 cells, we normalized their expression to an arbitrary value of 0.10 for quantification purposes. Error bars represent mean ± standard deviation (SD).

Fig 4: Nuclear HK2 modulates chromatin accessibility and is involved in the maintenance of DNA integrity.a, Proteins that interact with NLS1–HK2, as determined by BioID coupled with mass spectrometry; n = 3 biological repeats. b, Intensity of the PLA signal of endogenous HK2 and MAX, SIRT1, IWS1, CTR9 and SPIN1 in NB4 cells. A.u., arbitrary units; n = 278 cells from three biological repeats. c, Chromatin accessibility, measured through ATAC-seq, following overexpression of NLS1–HK2 in NB4 cells; n = 3 biological repeats. d, LSC+ and LSC- signatures in EV control NB4 cells. LSC-: minimum, 5.15; maximum, 12.81; and median, 8.27. LSC+: minimum, 5.990; maximum, 12.030; and median, 9.325. e, LSC+ and LSC- signatures in NLS1–HK2 NB4 cells. LSC-: minimum, 6.840; maximum, 13.010; and median, 9.020. LSC+: minimum, 7.300; maximum, 12.340; and median, 9.895. f, ATAC-seq pathway enrichment analysis in EV control and NLS1–HK2 NB4 cells. The size of the pie chart slices is proportional to the FDR score, -log10(FDR), for each of the gene lists. Blue and pink lines pinpoint to pathways that overlap significantly with the HSC and granulocyte (GRAN) gene lists at FDR < 0.05 according to a Fisher’s exact test. g, Enhanced enrichment of the pathways in NLS1–HK2 and NLS2–HK2 ChIP–seq compared with EV control ChIP–seq in NB4 cells. h, Consensus motif identified by HOMER DNA-binding-motif analysis significantly enriched in NLS1– and NLS2–HK2 peaks at FDR < 0.05. The P value shows significant enrichment of bHLH motifs, determined using a Fisher’s exact test. The boxes with the dashed lines represent sequence similarity overlap. i, Enrichment of the consensus motif CACGTG in sequences of peaks associated with the selected pathways. The hypergeometric P value estimated using the Fisher’s exact test indicate the significance of the enrichment of the motif when compared with random sequences. c–e, Statistical analyses were performed using a two-tailed unpaired Student’s t-test. Data represent the mean ± s.e.m. d,e, In the box-and-whisker plots, the horizontal lines mark the median, the box limits indicate the 25th and 75th percentiles, and the whiskers extend to 1.5× the interquartile range from the 25th and 75th percentiles.Source data

Fig 5: Effects of siIws1 or siSupt6 on mRNA splicing and export in mouse embryos. (A) Zygotes were electroporated with siControl, siIws1 or siSupt6 and cultured to the blastocyst stage. At the indicated times, splicing speckles were detected using anti-SC35. At least 15 embryos were used for each group. The fluorescence intensity of SC35 was measured in each specimen; mean values are depicted in the graph. (B) The pre-mRNA splicings of Cdk9 and c-Myc are defective in siIws1 and siSupt6 embryos. The levels of spliced and unspliced intron 1 were quantified by RT-qPCR. The expression level of the 18 S rRNA in siControl was set as 1 and used as an internal control. (C) Bulk Poly(A)+ mRNA accumulation in siIws1 or siSupt6 embryos. Poly (A)+ mRNAs were labeled by FISH using a specific oligo-dT probe and analyzed by confocal microscopy. The fluorescence intensities are depicted in the graph. At least 15 embryos were analyzed for each stage. (D) Iws1 colocalizes and interacts with Alyref. Four-cell embryos were double-immunostained against Iws1 and Alyref. PLA shows the putative interaction of the two factors. DNA was counterstained with DAPI. (E) Four-cell stage siControl, siIws1, and siSupt6 embryos were double-stained for Alyref and Pol II CTD Ser2p. PLA shows that either Iws1 or Supr6 is required for the interaction of Alyref with Pol II CTD Ser2p. Scale bars: 20 µm.