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. 2018 Oct 20;14(13):1883–1891. doi: 10.7150/ijbs.27854

Figure 4.

Figure 4

Shikonin induced necroptosis in T24 resistant BC cells. (A) T24 resistant cells were treated with 0.4 μM shikonin (SKN) and 25 µM cisplatin (Cis) alone or their combination (C+S) for 24 h. The expression of p-RIP3 and RIP3 was analyzed by western blot. (B) T24 resistant cells were pretreated with RIP3 inhibitor (GSK872, 2 µM) or MLKL inhibitor (NSA, 2 µM) for 1 h, and treated with 0.4 μM shikonin and 25 µM cisplatin alone or their combination for another 24 h. The cell viability was analyzed by MTT assay. (C) T24 resistant cells were pretreated with caspases inhibitor (z-VAD, 10 µM) for 1 h, and treated with 0.4 μM shikonin and 25 µM cisplatin alone or their combination for another 24 h. The cell viability was analyzed by MTT assay. (D) T24 resistant cells were transfected with RIP3 siRNA, and subsequently treated with 0.4 μM shikonin and 25 µM cisplatin alone or their combination for 24 h. The expression of RIP3 and p-MLKL was analyzed by western blot. (E) The T24 resistant cells were treated as in (D), the cell viability was analyzed by MTT assay. (F and G) T24 parental cells were treated as in (A). F, the cell viability was analyzed by MTT assay. G, the apoptosis was analyzed by Hoechst 33258 staining. N, p>0.05; *, p<0.05; **, p<0.01; ***, p<0.001.