FIG. 3.

Experimental design for evaluating the phospho-Smc1 response in cycling and quiescent primary human PBMC. Panel A: Blood was collected by phlebotomy from two healthy donors at three different times over a 1-month period. For each sample, seven independent aliquots of blood were prepared as follows: Three 10-ml aliquots were used for technical replicates to examine the response of cycling human PBMCs (shown in the left, “in vitro” arm). PBMCs were isolated by Ficoll gradient, and the cells were placed in culture and activated with anti-CD3/28 antibodies plus IL-2. Cells were cultured for 8 days and then split and grown for an additional 2 days. Cells were either mock-irradiated (0 Gy) or irradiated and returned to the incubator. Cells were harvested at 2, 8 and 24 h after irradiation, and protein lysates were prepared from the cells and evaluated by ELISA in duplicate on two independent ELISA plates (results are shown in Fig. 4A and Supplementary Table 1). In parallel, four 5-ml aliquots were used to examine the response of noncycling human PBMCs (shown in the right, “ex vivo” arm). Two of the blood aliquots were mock-irradiated (0 Gy) and two aliquots were exposed to 5 Gy, and blood was incubated for 2 h. PBMCs were isolated by Ficoll gradient, and protein lysates were prepared from the cells and evaluated by ELISA in duplicate on two independent ELISA plates (results are shown in Fig. 4B and Supplementary Table 1). Panel B: In vitro exposures. Each blood draw was divided into three technical replicates. PBMCs were isolated by Ficoll gradient and the cells were placed in culture and activated with anti-CD3/28 antibodies plus IL-2. Cells were cultured for 8 days and then split and grown for an additional 2 days. Cells were either mock-irradiated (0 Gy) or irradiated. Cells were harvested 2, 8 and 24 h after exposure, and protein lysates were prepared and evaluated by ELISA in duplicate. The mean concentrations of p-Smc1(pS957) and p-Smc1(pS966) were calculated from the standard peptide curve and the values were normalized to cell count. The means ± SD of the values of all measurements for all technical replicates from all three blood draws are plotted. Panel C. Ex vivo exposures. For each of the same three blood draws described in panel A, four additional aliquots of whole blood were set up. Two were mock-irradiated (0 Gy) and two were exposed to 5 Gy. PBMCs were isolated by Ficoll gradient at 2 h after exposure, protein lysates were prepared, and phospho-protein concentrations were measured by ELISA in duplicate. The mean concentrations of p-Smc1(pS957) and p-Smc1(pS966) were calculated from the standard peptide curves, and the values were normalized to cell count. The mean concentration ± SD for all technical replicates from the three blood draws is plotted.