Figure 6

Activation of DDR in vitro and in vivo and its inhibition by chromatin neutralizing/degrading agents. (a) Time course of activation of H2AX in NIH3T3 cells following co-cultivation with dead Jurkat cells as detected by indirect immunofluorescence. The experiment was done in duplicate at each time point and 100 cells were analyzed in each case and the average mean fluorescence intensity (MFI) values are depicted in the graph. (b) Prevention of H2AX activation in NIH3T3 cells by CNPs, DNase I and R-Cu following co-cultivation with dead Jurkat cells at 6 h. The experiment was done in duplicate and 500 cells were analyzed in each case for calculating MFI. The histograms depict mean (±S.E.) values in each case. Results were analysed by Student’s t-test. ****P<0.0001. Note that live Jurkat cells do not activate H2AX. (c) Activation of various DDR and DNA repair proteins following co-cultivation of NIH3T3 cells with dead and live Jurkat cells at 6 h as detected by indirect immunofluorescence. The experiments were done in duplicate and 500 cells were analyzed for calculating MFI. The histograms depict mean (±S.E.) values in each case; results were analyzed by Student’s t-test. ****P<0.0001. (d) Activation of H2AX in vital organs and PBMCs following i.v. injection of dead and live B16-F10 cells into mice and their prevention by CNPs, DNase I and R-Cu. One hundred thousand B16-F10 cells were injected i.v. and animals killed after 24 h. The experiments were done in duplicate and 1000 cells were analyzed in each case for calculating MFI. The histograms depict mean (±S.E.) values in each case; results were analyzed by Student’s t-test. *P<0.05, **P<0.01.