Mutation of AAD impairs ATR-Chk1 pathway <i>in vivo</i>.

<p>(<b>A</b>) Immunostaining of RPA (red and upper panel) in <i>TopBP1^ki/+</i> cells 36 hr after transfection with GFP-shLuc or GFP-shTop2 without or with the indicated treatment. ATRi, ATR inhibitor. GFP-shLuc or GFP-shTop2 transfection is visualized by GFP (green). DNA was stained with DAPI (blue). Inset shows the enlargement of selected areas. (<b>B</b>) Immunostaining of phosphorylation of Chk1-S317 (pChk1, red, upper panel) in <i>TopBP1^ki/+</i> cells 36 hr after transfection with GFP-shLuc or GFP-shTop2 without or with the indicated treatment. ATRi, ATR inhibitor. GFP-shLuc or GFP-shTop2 transfection is visualized by GFP (green). DNA was stained with DAPI (blue). GFP-shChk1 transfection (right panel, arrows) served as a negative control for pChk1 staining. (<b>C</b>) Quantification of fluorescent density of phosphor Chk1-S317 staining (pChk1) of indicated samples from B. The data represent the mean ± SD of at least 200 cells (or GFP positive cells) and were repeated three times. One-way ANOVA pair-test was performed for the statistical analysis. ***<i>P</i><0.001; <i>n.s.</i>, not significant.