Figure 2. Recruitment of Dicer to DNA double-strand breaks in AsiSI-ER U2OS cells. (A) Structure of genomic loci assessed by quantitative RT-PCT (left) and genome-wide AsiSI-ER target site distribution; n, number of predicted AsiSI-ER target sites (right); fwd/rev, forward/reverse. (B) Immunoblots detecting total Dicer (A-2) and γH2A.X after induction of DNA double-strand breaks (DSBs). 4OHT, 4-hydroxytamoxifen. Immunoblots were quantified using ImageJ. (C) Confocal imaging of phosphorylated Dicer (p-DCR-1) and γH2A.X (top). All quantifications represent number of cells exhibiting shown phenotype. Quantification using ImageJ RGB profiler (bottom). (D) ChIP analysis showing Dicer occupancy at DSBs DS1/2 in wild-type and AsiSI-ER U2OS cells using site-specific primers. GAPDH, control locus. *, P < 0.05; error bars, means ± SEM of three biological replicates. (E) ChIP analysis showing Dicer occupancy at DS1 in absence or presence of recombinant RNase III preincubation. *, P < 0.05; error bars, means ± SEM of three biological replicates. (F) ChIP-seq signal upon +4OHT incubation at 200 γH2A.X-positive/negative genic sites after removal of duplicate reads. A rolling mean of 1 kb was applied after removal of 2% of the top and bottom values. Shadow, rolling SD. (G) Snapshot showing Dicer binding at genic AsiSI target site upstream of TRIM37 before (4OHT−) and after (4OHT+) DNA damage. Red box, proximal region to AsiSI site.
Figure 2.

Recruitment of Dicer to DNA double-strand breaks in AsiSI-ER U2OS cells. (A) Structure of genomic loci assessed by quantitative RT-PCT (left) and genome-wide AsiSI-ER target site distribution; n, number of predicted AsiSI-ER target sites (right); fwd/rev, forward/reverse. (B) Immunoblots detecting total Dicer (A-2) and γH2A.X after induction of DNA double-strand breaks (DSBs). 4OHT, 4-hydroxytamoxifen. Immunoblots were quantified using ImageJ. (C) Confocal imaging of phosphorylated Dicer (p-DCR-1) and γH2A.X (top). All quantifications represent number of cells exhibiting shown phenotype. Quantification using ImageJ RGB profiler (bottom). (D) ChIP analysis showing Dicer occupancy at DSBs DS1/2 in wild-type and AsiSI-ER U2OS cells using site-specific primers. GAPDH, control locus. *, P < 0.05; error bars, means ± SEM of three biological replicates. (E) ChIP analysis showing Dicer occupancy at DS1 in absence or presence of recombinant RNase III preincubation. *, P < 0.05; error bars, means ± SEM of three biological replicates. (F) ChIP-seq signal upon +4OHT incubation at 200 γH2A.X-positive/negative genic sites after removal of duplicate reads. A rolling mean of 1 kb was applied after removal of 2% of the top and bottom values. Shadow, rolling SD. (G) Snapshot showing Dicer binding at genic AsiSI target site upstream of TRIM37 before (4OHT−) and after (4OHT+) DNA damage. Red box, proximal region to AsiSI site.

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