Figure 2. Shikonin-induced necroptosis is dependent on activation of RIPK1/RIPK3/MLKL signaling. (A) Determination by qRT-PCR of RIPK1, RIPK3 and MLKL expression in CML cells treated with or without 20 mM shikonin for 45 min. Gene expression values were normalized to those of GAPDH. Data represent the mean ± SD of three independent experiments. **p < 0.01. (B, C) Western blot analysis of both total and phosphorylated RIPK1, RIPK3, and MLKL expression. CML cells were treated with increasing shikonin concentrations (0, 5, 10, or 20 μM) for 45 min without (B) or with (C) prior exposure to 50 μM Nec-1 or 35 μM zVAD-fmk. (D) Apoptosis assessment. CML cells were exposed to 20 μM shikonin for 1.5 or 3 h. Cells treated with 20 nM HHT for 24 h were used as positive control. Apoptosis was measured by a colorimetric assay to detect caspase-3 activation. Data represent the mean ± SD of three independent experiments. **p < 0.01.