Research Paper Volume 15, Issue 24 pp 14900—14914

Figure 6. Acetylshikonin promoted cell death via necroptotic RIPK1, RIPK3, and MLKL signaling activation. (A) Fluorescence microscope images showing MLKL phosphorylation in H1299 and A549 cells following incubation with acetylshikonin (2.5 μM) for 0–4 h. Scale bar = 50 nm. (B, C) Western blot analysis showing RIPK1, RIPK3, and MLKL protein phosphorylation levels in NSCLC cells treated with acetylshikonin (2.5 μM) for 0–4 h (n = 4). (D) Fluorescence microscope images showing MLKL phosphorylation in H1299 and A549 cells preincubated with necrostatin-1 (20 nM) and 7-Cl-O-Nec-1 (30 nM) for 1 h and then incubated with acetylshikonin (2.5 μM) for a further 4 h. Scale bar = 50 nm. (E, F) CCK-8 assays indicating the viability of H1299 and A549 cells preincubated with necrostatin-1 (20 nM) and 7-Cl-O-Nec-1 (30 nM) for 1 h and then incubated with acetylshikonin (2.5 μM) for a further 24 h (n = 4). Untreated cells were used as controls. Results are shown as means ± SD. ^*p < 0.05 compared to untreated control. ^#p < 0.05 compared to acetylshikonin alone group.