Skip to main content
. 2021 Feb 26;40(7):e106018. doi: 10.15252/embj.2020106018

Figure EV4. Related to Fig 3. NBS1 co‐localization with DNA‐RNA hybrids in U2OS depleted of DDX5 or BRCA2 and DNA‐RNA hybrids, transcription levels, and DDX5 occupancy at different genomic locations in U2OS DIvA cells.

Figure EV4

  1. Left: Representative images of in situ PLA experiment performed between NBS1 and S9.6 antibodies in U2OS cells transfected with either siRNA control (siC) or siRNA specific for DDX5 (siDDX5). When indicated, cells were transfected with RNAseH1 (RH) 24 h before fixation. Single antibody controls from untreated cells are shown. Scale bar indicates 10 µm. Nuclei as defined by auto threshold plugin on the DAPI image (ImageJ) are outlined in yellow. Right: Quantification of the number of PLA spots per nucleus in different conditions, as indicated. The data represent at least 400 cells per condition from three independent experiments. For statistical comparison of the differences between the samples, we applied a Kruskal–Wallis test followed by Dunn’s multiple comparison test and the P‐values show significant differences. The red line in the plot indicates the median, and each symbol represents a single PLA spot.
  2. DRIP‐qPCR signal values at RBMXL1, ASXL1, HIST1H2BG, WDR90, and SNPRN loci in U2OS DIvA cells transfected with the indicated siRNAs and treated in vitro with RNase H1 (RH) pre‐immunoprecipitation where indicated. The experiment was performed in both untreated cells (−OHT) and after tamoxifen addition (+OHT). The data represent the mean ± SEM from at least four independent experiments. The statistical significance of the difference was calculated with unpaired one‐tailed Student t‐test, and the P‐values show the significant differences.
  3. Relative RBMXL1, ASXL1, HIST1H2BG, WDR90, and SNPRN gene expression levels in U2OS DiVA cells transfected with the indicated siRNAs. The data represent the mean ± SEM from two or three independent experiments. The statistical significance of the difference was calculated with unpaired Student t‐test; the P‐values show the significant differences.
  4. γH2AX (top) and DDX5 (bottom) ChIP‐qPCR signal values at RBMXL1, ASXL1, HIST1H2BG, WDR90, and SNPRN loci in U2OS DiVA cells transfected with the indicated siRNAs and either left untreated (−OHT) or treated with tamoxifen (+OHT). The green line represents the background levels of DDX5 signal. The data represent the mean ± SEM from at least three independent experiments. The statistical significance of the difference was calculated with unpaired one‐tailed Student t‐test, and the P‐values show the significant differences.