Fig. 6.

γH2AX hyper-activation of cleaved PARP or parthanatos, cleaved PARP and γH2AX or DNA Damage assay. Jurkat untreated, treated with 0.5 μM shikonin or pre-treated zVAD (20 μM) or Necrostatin-1 (60 μM) for 2 h then incubated with 0.5 μM shikonin for 24 h. After gating on live and dead cells from a Zombie NIR vs. Caspase-3-BV650 dot-plot untreated or treated live and dead Jurkat cells were analysed on a RIP3-PE v Caspase-3-BV650 dot-plot. From which early and late apoptotic, necroptotic, RIP1-dependent apoptotic and double negative (DN) populations were analysed for γH2AX and cleaved PARP, see Figs. 1S and 2S for detailed information. The incidence of γH2AX hyper-activation of cleaved PARP or parthanatos (a), cleaved PARP (b) and DNA Damage (c) were determined for all populations listed above. Average % ± SD, (n = 3) were analysed for significance by One Way ANOVA (P  < 0.5) and post ad hoc tested for significance, NS (P > 0.5, not significant), significance was P < 0.05*, P < 0.01**, P < 0.001***, P < 0.0001**** compared to untreated cells and between treatments as indicated