Figure 4: Shikonin inhibits cancer cell migration and affects microtubule structure and dynamics. (a) Typical pictures at 0, 6, and 12 h of scratch migration assays using SK-BR-3 cells treated with different effective, but in this time-frame subtoxic, concentrations of shikonin. (b) Statistical quantification of the scratch migration assay. Data points represent the mean SEM of at least three independent experiments. (c) Live imaging of U2OS-GFP-αTubulin cells stably transfected with a GFP fusion construct of α-tubulin and treated with 25 μM shikonin. With increasing cellular concentrations of shikonin, the number of distinct tubulin filaments decreased and the tubulin staining became progressively diffuse. (d) Live imaging of RPE-1-GFP-EB3 cells stably expressing GFP-EB3 and treated with 25 μM shikonin. Shikonin caused a slowdown and finally a complete disappearance of EB3 particles within 3min after application, indicating disrupted microtubule formation (*significant difference according to Student’s -test, ).