Figure 5. Prerequisite of Dicer S1728/1852 phosphorylation for damage-induced dsRNA processing in HEK293 cells. (A) Confocal imaging of RFP-tagged Dicer constructs and dsRNA (J2) in Dicer-depleted (Dicer KD) cells transfected with RFP-Dicer constructs in the absence or presence of Leptomycin B (LMB). n > 30. Control, mock transfected Dicer KD cells. See also Fig. S5 F for additional controls. All quantifications represent the number of cells that have the shown phenotype. (B) Quantification of dsRNA from A using ImageJ. J2 signal was normalized to the background. *, P < 0.05; error bars, means ± SEM of three biological replicates. (C) Immunoblots detecting ataxia telangiectasia mutated (ATM) or phospho-ATM (S1981) and γH2A.X in the absence or presence of AsiSI-ER expression vector pBABE::AsiSI-ER in wild-type HEK293 cells. (D) Confocal imaging of 53BP1 and γH2A.X in wild-type cells transfected with recombinant AsiSI-ER expression vector pBABE::AsiSI-ER. All quantifications represent the number of cells that have the shown phenotype. (E) Confocal imaging of RFP-tagged Dicer constructs and dsRNA (J2) in Dicer-depleted (Dicer KD) cells cotransfected with RFP-Dicer constructs and pBABE::AsiSI-ER. Controls, mock transfected wild-type or Dicer KD cells. All quantifications represent the number of cells that have the shown phenotype. (F) Quantification of dsRNA, *, P < 0.05; error bars, means ± SEM of three biological replicates; from E using ImageJ. J2 signal normalized to background. (G) Immunoblots detecting expression of RFP-Dicer constructs (RFP) in wild-type and Dicer-depleted HEK293 cells cotransfected with pBABE::AsiSI-ER. Dox., doxycycline; Rad21, loading control; #, unspecific signal.
Figure 5.

Prerequisite of Dicer S1728/1852 phosphorylation for damage-induced dsRNA processing in HEK293 cells. (A) Confocal imaging of RFP-tagged Dicer constructs and dsRNA (J2) in Dicer-depleted (Dicer KD) cells transfected with RFP-Dicer constructs in the absence or presence of Leptomycin B (LMB). n > 30. Control, mock transfected Dicer KD cells. See also Fig. S5 F for additional controls. All quantifications represent the number of cells that have the shown phenotype. (B) Quantification of dsRNA from A using ImageJ. J2 signal was normalized to the background. *, P < 0.05; error bars, means ± SEM of three biological replicates. (C) Immunoblots detecting ataxia telangiectasia mutated (ATM) or phospho-ATM (S1981) and γH2A.X in the absence or presence of AsiSI-ER expression vector pBABE::AsiSI-ER in wild-type HEK293 cells. (D) Confocal imaging of 53BP1 and γH2A.X in wild-type cells transfected with recombinant AsiSI-ER expression vector pBABE::AsiSI-ER. All quantifications represent the number of cells that have the shown phenotype. (E) Confocal imaging of RFP-tagged Dicer constructs and dsRNA (J2) in Dicer-depleted (Dicer KD) cells cotransfected with RFP-Dicer constructs and pBABE::AsiSI-ER. Controls, mock transfected wild-type or Dicer KD cells. All quantifications represent the number of cells that have the shown phenotype. (F) Quantification of dsRNA, *, P < 0.05; error bars, means ± SEM of three biological replicates; from E using ImageJ. J2 signal normalized to background. (G) Immunoblots detecting expression of RFP-Dicer constructs (RFP) in wild-type and Dicer-depleted HEK293 cells cotransfected with pBABE::AsiSI-ER. Dox., doxycycline; Rad21, loading control; #, unspecific signal.

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