Analysis of Residual DSBs in Ataxia-Telangiectasia Lymphoblast Cells Initiating Apoptosis

Figure 3

(a) Immunodetection of γH2AX in lymphoblasts. DSBs were scored by γH2AX foci detection in TUNEL-negative, An+/PI−, and An+/PI+ cells. Pan-nuclear γH2AX staining was scored in TUNEL-negative, An+/PI−, and An+/PI+ and in TUNEL-positive cells. (b) γH2AX-labeling in viable (TUNEL-negative) cells. The number and frequency of viable cells with γH2AX foci are reflected in the bars. Within this fraction, the frequency of cells with <10 foci or with ≥10 foci is shown inside the bars. The asterisks indicate statistical differences between normal and AT lymphoblasts in the fraction of cells with γH2AX foci or in the fraction of cells with ≥10 γH2AX foci ( test; values from to ). The frequencies for each category are calculated over the total number of TUNEL-negative scored cells. A minimal number of 350 TUNEL-negative cells were analyzed for each cell type and each time point. The apoptotic rate measured with TUNEL is depicted in the graph as a continuous line. Values for TUNEL-positive cells are given under the -axis and are those corresponding to Figure 1(a). (c) γH2AX-labeling in Annexin-positive cells. AT and normal cells were irradiated and fractions corresponding to EA and LA were isolated by cell sorting. An+/PI− and An+/PI+ cells were classified into those with or without γH2AX foci and those with pan-nuclear γH2AX staining. The frequency of cells with γH2AX foci is depicted next to the bar. Within this fraction, the frequency of cells with less than 10 foci (light pink) or with 10 or more foci (pink) is shown inside the bars. The asterisks indicate statistical differences between normal and AT lymphoblasts in the frequency of cells with ≥10 γH2AX foci ( test; values from to ). The frequencies are calculated over the total number of An+/PI− and An+/PI+ sorted cells. A minimal number of 400 cells were analyzed for each cell type and each time point.

(a)

(b)

(c)