Fig. 4.

Hyper-activation of PARP, parthanatos and DDR assay. Jurkat untreated, treated with 0.5 µM shikonin or pre-treated zVAD (20 µM) and 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/resting, RIP1-dependent apoptotic and double negative (DN) populations were analysed for H2AX and PARP, see Figs. 1S, 2S for detailed information. The incidence of hyper-activation of PARP (a), parthanatos (b) and DDR (c) were determined for all populations listed above. n = 3, student t test NS (not significant), P < 0.05*, P < 0.01**, P < 0.001*** compared to untreated cells