Research Paper Volume 12, Issue 17 pp 17662—17680

Figure 3. Shikonin induces ROS production and MMP loss in CML cells. (A, B) Assessment of ROS generation by H₂DCFDA staining. K562 and 32Dp210-T315I cells were pretreated with or without Nec-1 (50 μM) or zVAD-fmk (35 μM) for 1 h and then exposed to 20 μM shikonin for 1 h. Upper row images in (A) are phase contrast micrographs, and lower row images are fluorescence micrographs. Green signal indicates ROS generation as detected by H₂DCFDA staining. Quantitation of DCF fluorescence from flow cytometry assays is shown in (B). Values are expressed as the mean ± SD of 3 experiments. **p < 0.01. (C) Analysis of MMP by JC-1 staining. Control, Nec-1 (50 μM), or zVAD-fmk (35 μM) pre-treated cells were exposed to 20 μM shikonin for 1 h, stained with JC-1 dye, and analyzed by flow cytometry. Results represent data from three independent experiments. (D) Western blot analysis of total and phosphorylated BCR/ABL expression in CML cells treated with 5-20 μM shikonin for 3 h.