FIGURE 6.

Rapamycin treatment partially rescues the migration and CDC42/RAC1 activation defects in TSC2 knockdown colon cancer cells. A, KM20 control (Con) and TSC2 knockdown cells were treated with dimethyl sulfoxide or rapamycin (20 nm) for 5 h and analyzed for CDC42 and RAC1 activation using GST-PBD pulldown assays. The amount of activated and total CDC42 and RAC1 in the cells were detected using the anti-CDC42 and anti-RAC1 antibodies on Western blots. The GST-PBD bound panels represent the activated fraction of CDC42 and RAC1, and 10% of cell lysates were used to show the total expression of CDC42 and RAC1 (Lysate panels). The expression of TSC2, p-AKT (phospho-Ser⁴⁷³), total AKT, and tubulin are detected in the cell lysates. The percentage of activation was quantified by normalizing the level of activated CDC42 and RAC1 (Bound panels) to total proteins in the lysates (Lysate panels), and indicated by the numbers below the representative panels. B, KM20 control and TSC2 knockdown cells were subjected to trans-well migration analysis in the presence or absence of rapamycin in the lower chamber. The numbers of migrated cells were quantified, normalized to the control cells, and expressed as percentage of migration. Data represent mean ± S.E. (n = 3; *, p < 0.05). Rapa, rapamycin.