Inhibition of HDAC6 activity rescues invasion, 3D motility, polarized MT acetylation, and cell morphology in paxillin-depleted cells. (A and B) Montages (A) and quantitation (B) of MDA-MB-231 cell invasion into collagen- and fibronectin-rich 3D gels. n = 4 individual experiments. (C–E) Migration tracks (C), velocity (D), and directionality analysis (E) of MDA-MB-231 cell migration through 3D CDMs. A minimum of 30 tracks from three individual experiments was used for 3D migration analyses. (F and G) Western blots (F) and images (G) showing a rescue of α-tubulin acetylation in cells treated with paxillin RNAi and 2 µM tubacin during 4 h of 3D migration. Arrows indicate direction of migration, and boxed regions indicate the cropped region used for the pseudocolored acetylated (Ac) α-tubulin lookup table (LUT) image. (H) Representative kymographs from 3D CDM migration time-lapse videos of paxillin-depleted MDA-MB-231 cells treated with vehicle or 2 µM tubacin at t = 0. Data are represented as the mean ± SEM. *, P < 0.05; **, P < 0.005; ***, P < 0.0005.