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. 2023 Mar 16;18(4):952–968. doi: 10.1016/j.stemcr.2023.02.003

Figure 6.

Figure 6

Loss of TDP-43 or DDX5 induces R-loop accumulation and DNA damage

(A) Immunostaining showed that TDP-43 KD or DDX5 KD induced the R-loop formation in mESCs. Re-expression of TDP-43 (TDP-43-rescue), DDX5 (DDX5-rescue), or RNase H1 OE in KD cells rescued the defect. Scale bar, 20 μm.

(B) Quantification of R-loop immunostaining intensity in (A). At least 20 visual fields containing 500 cells were analyzed in three independent experiments.

(C) Dot-blot analysis on genomic DNA confirmed that KD of TDP-43 or DDX5 increased R-loop levels in mESCs. Re-expression of TDP-43 or DDX5, or RNase H1 OE in KD cells rescued the defect.

(D) Quantification of dot-blot intensity in (C), with dsDNA as internal reference.

(E) CIdU incorporation assay showed that KD of TDP-43 or DDX5 increased the level of ssDNA monitored by CIdU (red). Re-expression of TDP-43 or DDX5, or RNase H1 OE in KD cells rescued the defect. Scale bars, 20 μm.

(F) Quantification of immunostaining intensity in (E). At least 20 visual fields containing 1,000 cells were analyzed in each group.

(G) Neutral comet assay revealed that KD of TDP-43 or DDX5 increased DNA DSBs level. Re-expression of TDP-43 or DDX5, or RNase H1 OE in KD cells rescued the defect. Left panel shows the quantification of comet tail length. Right panel shows representative images. At least 200 tails were analyzed in each group. Scale bar, 400 μm.

(H) More DDX5 KD and TDP-43 KD mESCs contained micronuclei. Left panel shows quantification result. Right panel shows the representative image of micronucleus (arrow). At least 50 visual fields containing 1,000 cells were analyzed in each group. Scale bars, 10 μm.

(I) DDX5 KD and TDP-43 KD mESCs displayed higher rate of aneuploidy. Left panel shows quantification result. Right panel shows representative images. At least 150 metaphase spreads were examined in three replications in each group. Scale bars, 20 μm. All experiments were independently repeated three times with similar results. Data are shown as mean ± SD, two-tailed Student’s t test.