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Figure 5: Effect of B2 on migration and colony formation of PC3 cells. For migration assay, a straight scratch wound was created using a 200 μL micropipette tip. The cells were treated with B2 (12, 25, and 50 μg/mL). DMSO (0.1%) and betulinic acid were used as negative and positive controls, respectively. The wounds were photographed using EVOS f1 digital microscope at zero, 6, and 12 h. (a) The images taken form 0.1% DMSO treated cells showed completely closed wound due to the successful migration of PC3 cells within 12 h, whereas in B2-treated group, the wound remained open even after 12 h period time. B2 (12 μg/mL) caused significant inhibition of PC3 cell migration at subcytotoxic dose. (b) Graph illustrates the time- and dose-dependent inhibitory effect of B2 on migration of PC3 cells. The distance of cell-free area was measured using Leica Quin software, and the results are presented as mean percentage of migration inhibition ± SD in comparison with the negative control. (c) Inhibitory effect of B2 on survival of PC3 colonies in colony formation assay. The picture clearly depicts the dose-dependent inhibition of PC3 colonies. PC3 cells (500 cells/well) were seeded in 6-well plate and treated with B2 (6, 12, 25, and 50 μg/mL) for 48 h. Betulinic acid (10 μg/mL) and 1% DMSO were used as positive and negative controls, respectively. The cells were maintained until sufficiently large colonies (≥50 cells) were produced for 10 days. The colonies were fixed, stained with 0.2% crystal violet, and counted under stereomicroscope. (d) Graphical representation depicts percentage of plating efficiency of representative test groups. Plating efficiency was determined by the percent ratio of number of colonies developed to the number of cells initially seeded. (e) Graphical representation illustrates the percentage of surviving fraction obtained after the treatment with B2. The percent surviving fraction of the PC3 colonies was decreased with increasing concentration B2. All the values are presented as mean ± SD (n = either 6 or 10), represents , and represents .