Increased p21 expression after treatment of MDA-MB-468 cells with microtubule-depolymerizing agents. (a) Cells were treated with 1 μM nocodazole, 100 nM paclitaxel, 100 nM vincristine, or 100 nM colchicine for 24 hours. Whole-cell lysates were subjected to SDS-PAGE and probed for p21. p53, which is mutated in these cells (41), served as a loading control. (b) Poly(A)⁺ RNA from cells treated with 1 μM nocodazole for various periods of time was analyzed by RT-PCR using primers specific for p21. β-actin served as a control. (c) Cells were treated with 1 μM nocodazole for various lengths of time before SDS-PAGE and immunoblotting for p21, GADD45 (GD45), or p53 (as a loading control). (d and e) Cells were treated with 1 μM nocodazole for 24 hours, fixed in 50% ethanol, stained with PI, and sorted into 2N and 4N populations by flow cytometry (gates set as shown). Protein (50 μg) from unsorted (total) or sorted (2N or 4N) cell populations was subjected to SDS-PAGE followed by immunoblotting. Cyclin B and cyclin E served as markers of the G₂ and G₁ populations, respectively, while PARP served as a loading control.