Fig. 4
From: Kinase-dead ATR differs from ATR loss by limiting the dynamic exchange of ATR and RPA
Atr+/KD cells display HU-induced genome instability and checkpoint defects. a Metaphases from B cells treated with HU (0.25 mM) or Aphidicolin (0.5 µM) were analyzed for chromatid breaks, chromosome breaks, and breaks in fusions. The bar graphs represent the means ± SD and unpaired two-tailed t test was used. Three (HU) or two (Aphidicolin) independent experiments were performed. A total of 142 (Atr+/+), 161 (Atr+/−), and 156 (Atr+/KD) metaphases in HU, and 83 (Atr+/+), 90 (Atr+/−), and 91 (Atr+/KD) in Aphidicolin were analyzed. b The means ± SD of the number of complex rearrangements (fusions, quadri-radials) in HU were plotted from three independent experiments. Unpaired two-tailed t test was used. c Atr+/+, Atr+/−, and Atr+/KD MEFs were pulse-labeled with BrdU for 30 min, washed and released in unchallenged media up to 6 h. Dot plots show the percentage of BrdU-positive cells and histograms show the cell cycle distribution of BrdU-positive cells. d Atr+/+, Atr+/−, and Atr+/KD MEFs were pulse-labeled with BrdU for 30 min, treated with 2 mM HU for 3 h and then released from HU in unchallenged media up to 12 h. Data were plotted as in (c). e Primary Atr+/+, Atr+/−, and Atr+/KD MEFs were treated with 2 mM HU for 3 h or 200 nM CPT for 2 h, with or without ATRi (10 µM, VE-821). Whole-cell extracts (WCE) were immunoblotted with the indicated antibodies. f Atr+/+, Atr+/−, and Atr+/KD MEFs were treated with increasing concentrations of HU (0.01, 0.05, 0.1, 0.2 mM) for 1 h, and WCE were immunoblotted with the indicated antibodies. g Histogram shows the quantification of pCHK1/CHK1 at 0.2 mM HU derived from three independent experiments. The means ± SD are shown and unpaired two-tailed t test was used. h Atr+/KD MEFs were treated with 0.2 mM HU for 1 h with or without pre-treatment for 1 h with the indicated checkpoint inhibitors. ATRi (VE-821) and CHK1i (LY603218) were used at 10 µM concentration, ATMi (KU-55933) at 15 µM, and DNA-PKcsi (NU7441) at 5 µM. WCE were immunoblotted with the indicated antibodies. Source data are provided as a Source Data file
