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

Figure 5.

Figure 5

Lnc530-TDP-43-DDX5 complex is localized on R-loop under transcription stress condition

(A) S9.6 IP showed that TDP-43 and DDX5 were accumulated on R-loops in mESCs under transcription stress condition. RNase H1 over-expression (OE) or Lnc530 KD reduced the accumulation of TDP-43 and DDX5 on R-loops.

(B) S9.6 IP combined with RT-qPCR analysis for detection of Lnc530 on R-loops in mESCs and NIH3T3. Left panel shows the relative levels of Lnc530. Right panel shows the RT-PCR bands. Actb was set as internal reference.

(C) Immunostaining showed that Lnc530 KD impaired the accumulation of TDP-43 (left) or DDX5 (right) on R-loops. Scale bars, 20 μm.

(D) S9.6 IP showed that TDP-43 KD impaired the allocation of DDX5 on R-loops. Re-expression of TDP-43 (TDP-43-rescue) rescued the defect.

(E) Immunostaining showed that accumulation of Lnc530 (left) or DDX5 (right) on R-loops was impaired in TDP-43 KD mESCs. Re-expression of TDP-43 (TDP-43-rescue) rescued the defect. Scale bars, 20 μm.

(F) S9.6 IP showed that DDX5 KD impaired the association of TDP-43 with R-loops, which was restored by re-expression of DDX5 (DDX5-rescue).

(G) Immunostaining revealed that accumulation of Lnc530 (left) and TDP-43 (right) on R-loops was impaired in DDX5 KD mESCs, and the defects were rescued by re-expression of DDX5 (DDX5-rescue). Scale bars, 20 μm. All experiments were independently repeated three times with similar results. The relative protein levels in (D) and (F) were normalized by input.