Fig. 2: ATR kinase activity is essential for productive DNA replication.
a Flow cytometry profiles of CD21-Cre+ Atr+/C, AtrC/-, and AtrC/KD B cells pulse-labeled with BrdU for 30 min at 48 h post-stimulation. Representative dot plots are shown. Histogram plots of S-phase cells are shown with the separation in BrdU-negative (BrdU−) and BrdU-positive (Brdu+) cells. Quantification of the percentage of BrdU negative cells from four independent experiments is reported, together with the two-tailed t-test statistical analysis. Data are presented as mean values ±SEM. b CD21-Cre+ Atr+/C, AtrC/-, and AtrC/KD B cells were labeled with CldU for 30 min and with IdU for an additional 30 min on day 2 of CSR, and DNA fibers were spread and stained. The length of IdU fibers was measured using Image J and plotted as shown. Two-sided Mann-Whitney test was used. Data are presented as mean values ±SD. c CD21-Cre+ Atr+/C, AtrC/-, and AtrC/KD B cells at 48 h post-stimulation were stained for γH2AX and PI and analyzed by flow cytometry. Quantification of three independent experiments of γH2AX/PI staining is shown for CD21-Cre+ Atr+/C and AtrC/- B cells, with two-tailed t-test analysis indicated. Data are presented as mean values ±SEM. d Alkaline comet assay was performed in CD21-Cre+ Atr+/C AtrC/- and AtrC/KD B cells at 48 h post-stimulation. One representative of two independent experiments is shown. The tail moment (arbitrary units, a.u.) of single cells is reported with two-taled t-test analysis indicated. Data are presented as mean values ±SD. e CD21-Cre+ Atr+/C, AtrC/-, and AtrC/KD B cells were lysed on day 2 of CSR, and the indicated proteins were analyzed by western blot. f 48 h stimulated WT B cells were left untreated, treated with 5 µM ATRi (VE-821) from the time of B cells activation or with 10 µM ATRi (VE-821) for 2 h right before BrdU labeling (30 min) and collection (immediately after labeling). Source data are provided as a Source Data file.
