Figure 7. Impaired recruitment of DNA repair factors upon mutation of Dicer in A549 cells. (A) Immunoblots (top) and confocal microscopy (bottom) detecting endogenous Dicer (H212, A-2, 13D6) in wild-type and Dicer knockout (ΔDicer) A549 cells. Ponc., Ponceau S, loading control; #, unspecific signal. (B) Immunoblots detecting γH2A.X levels in wild-type A549 cells after transfection with pBABE::AsiSI-ER plasmid and 4OHT incubation (2 h pulse). (C and D) Confocal imaging of MDC1 (C) and 53BP1 (D) in wild-type A549 cells after transfection with pBABE::AsiSI-ER plasmid and 4OHT incubation as indicated. (E and F) Imaging as in C and D, but performed in ΔDicer cells, including transfection of RFP-Dicer constructs. All quantifications represent the percentage of foci positive cells, n = number of cells analyzed. *, P < 0.05; error bars, means ± SEM of three biological replicates. (G) Immunoblots detecting expression of RFP-Dicer constructs in the absence or presence of 4OHT. (H) Model for DNA damage-induced redistribution of the cellular Dicer pool. In undamaged cells (control), Dicer is a predominantly a cytoplasmic protein that shuttles to the nucleus sporadically and is rapidly exported back to the cytoplasm (CP). In the presence of DSBs, the DNA damage response (DDR) targets a small fraction of the cellular Dicer pool by arguably sequential phosphorylation of serine residues S1016 (green) and S1728/S1852 (blue), which causes accumulation in the nucleoplasm (NP) and recruitment to DSBs. Phosphorylated Dicer (p-Dicer) binds and processes dsRNA, which may be produced by RNAPII transcription at lesions to promote the DDR. Phosphorylation of Dicer at S1016 may also alter the import/export rate.
Figure 7.

Impaired recruitment of DNA repair factors upon mutation of Dicer in A549 cells. (A) Immunoblots (top) and confocal microscopy (bottom) detecting endogenous Dicer (H212, A-2, 13D6) in wild-type and Dicer knockout (ΔDicer) A549 cells. Ponc., Ponceau S, loading control; #, unspecific signal. (B) Immunoblots detecting γH2A.X levels in wild-type A549 cells after transfection with pBABE::AsiSI-ER plasmid and 4OHT incubation (2 h pulse). (C and D) Confocal imaging of MDC1 (C) and 53BP1 (D) in wild-type A549 cells after transfection with pBABE::AsiSI-ER plasmid and 4OHT incubation as indicated. (E and F) Imaging as in C and D, but performed in ΔDicer cells, including transfection of RFP-Dicer constructs. All quantifications represent the percentage of foci positive cells, n = number of cells analyzed. *, P < 0.05; error bars, means ± SEM of three biological replicates. (G) Immunoblots detecting expression of RFP-Dicer constructs in the absence or presence of 4OHT. (H) Model for DNA damage-induced redistribution of the cellular Dicer pool. In undamaged cells (control), Dicer is a predominantly a cytoplasmic protein that shuttles to the nucleus sporadically and is rapidly exported back to the cytoplasm (CP). In the presence of DSBs, the DNA damage response (DDR) targets a small fraction of the cellular Dicer pool by arguably sequential phosphorylation of serine residues S1016 (green) and S1728/S1852 (blue), which causes accumulation in the nucleoplasm (NP) and recruitment to DSBs. Phosphorylated Dicer (p-Dicer) binds and processes dsRNA, which may be produced by RNAPII transcription at lesions to promote the DDR. Phosphorylation of Dicer at S1016 may also alter the import/export rate.

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