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. 2014 Jan 30;9(1):e87299. doi: 10.1371/journal.pone.0087299

Figure 2. B-cell mitogens activate the ATM signaling pathway in proliferating cells.

Figure 2

(A) Representative FACS plot of stimulated PBMCs showing the distribution of Cell Trace Violet stained cells versus CD19 staining for B cells. The indicated proliferating and non-proliferating populations were isolated by FACS sorting for subsequent analysis. (B) Immunofluorescence microscopy for γ-H2AX (red) of sorted non-proliferating (Non Prolif) or proliferating (Prolif) B cells. The DAPI stains for DNA are shown. (C) Untreated and 5 Gy γ-irradiated controls assayed as described in (B). (D) The average percentage of cells with γ-H2AX intensity >5X over background are plotted for three normal donors. Samples include B cells, untreated (0), 1 Gy, or 5 Gy γ-irradiation, sorted non proliferating (NP) and proliferating (P) cells for EBV, CpG, or αCD40/IL-4 treatments. Quantification is detailed in Figure S1. (E) Western blot analysis of Chk2, Chk2 phosphorylated on Thr68 as well as the control GAPDH. Untreated (Cntr) and 5 Gy irradiated (IR) Burkitt’s lymphoma cell line 2 (BL2) are included as a positive control. Numbers indicate the normalized densitometry value for phospho-Chk2 Thr 68.