Endogenous DNA damage and delayed repair in Dicer-depleted HEK293 cells. (A) Immunoblots detecting total Dicer (A-2) and γH2A.X levels after Dicer depletion (Dicer KD), expression of a scrambled shRNA (control KD) or reexpression of TAP-tagged Dicer (top). Quantitation of γH2A.X levels in wild-type and Dicer KD cells (bottom), *, P < 0.05; error bars, means ± SEM of three biological replicates. (B) Epifluorescence imaging of γH2A.X staining in wild-type and Dicer KD cells (top). Quantitation of γH2A.X levels as the percentage of γH2A.X-positive cells, n > 200 (bottom); *, P < 0.05; error bars, means ± SEM of three biological replicates. (C) Immunoblots detecting total Dicer (A-2), phospho-ATM (S1981), substrates of ATM/ATR phosphorylation, phospho-Chk1 (S345, S317), and γH2A.X after pulse-chase treatment with hydrogen peroxide (H₂O₂) in wild-type and Dicer KD cells (top). Quantitation of γH2A.X levels using ImageJ (bottom), *, P < 0.05; error bars, means ± SEM of three biological replicates.