Fig. 4: Coexistence of DIDO3, RNA pol II, DHX9, and R-loops, and alterations in DIDO3ΔE16 mutant ESC.

a BED peak files were imported to RStudio and annotated using the R/Bioconductor package ChIPseeker. Peak annotations are depicted as a percentage for HADIDO3, RNA pol II S2p, R-loops, and H3K4me3 (histone 3 trimethylated on lysine 4). b Co-immunoprecipitation of HA-tagged DIDO3, -common N-terminal region, or -DIDO3-exon16-specific C-terminal part (for constructs see [28]), and DHX9 helicase. c Co-immunoprecipitation of DIDO3 or DIDO3ΔE16 proteins and DHX9 helicase at R-loops precipitated with the S9.6 mAb, from sonicated chromatin of WT or DIDO3ΔE16 ESC. d Abundance of POU5f1 short and long 3′UTR after two or three applications of siDHX9 or siControl in WT and DIDO3ΔE16 ESC; β-ACTIN was used as control for equal RNA amounts. e Western blot analysis of DHX9 and CARM1 expression in WT and DIDO3ΔE16 ESC after two or three applications of siDHX9 or siControl; β-ACTIN was used as loading control. f Left: representative example of slot-blot analysis of restriction enzyme-digested genomic DNA, before and after immunoprecipitation with S9.6 mAb, alone or treated with RNaseH1 from WT or DIDO3ΔE16 ESC. Probes were used in duplicate; the first was developed with anti-ssDNA mAb and the second with anti-R-loop mAb S9.6. Right: quantification of slot–blot bands normalized to ssDNA values with ImageJ. Bars represent mean ± SEM. Statistical analysis was done with two-tailed Student’s t-test, **P ≤ 0.01; *P ≤ 0.05; DIDO3ΔE16, n = 3; WT, n = 3. g Western blot analysis of total lysates from WT and DIDO3ΔE16 ESC developed with anti-53BP1 and anti-phosphorylated CHK1 (pCHK1) antibodies; β-ACTIN was used as loading control. h Contour density plot of WT and DIDO3ΔE16 ESC after a 30-min EdU pulse, showing EdU incorporation versus DNA content. The percentages of events in cytometric gates defining different phases of the cell cycle are depicted.