Figure 2

PTC596 induces cell cycle arrest and apoptosis associated with the inhibition of microtubule polymerization. (A) Western blotting analysis of soluble and polymerized microtubules in MM cells detected using an anti-α-tubulin antibody. MM.1S were incubated in the presence or absence of PTC596 or paclitaxel for 4 h. After treatment, cell lysates were fractionated by centrifugation to separate free tubulin (soluble tubulin) from microtubules (polymerized tubulin). The graphs show the density volumes of α-tubulin normalized to that of the soluble fraction in the control. (B) Cell cycle analysis of MM.1S and H929 treated with the indicated doses of PTC596 for 24 h, exposed to BrdU for 2 h, followed by flow cytometric analyses. Data represent mean ± SD of triplicate experiments. *P < 0.05; ***P < 0.001; ns, not significant using one-way ANOVA. (C) Annexin V staining of MM.1S and OPM-2 treated with the indicated doses of PTC596 for 48 h. Apoptotic cells were detected as Annexin V-positive cells by flow cytometry. The representative flow cytometric profiles are shown in the left panels, and the results of duplicate experiments are shown in the right graphs. Data represent mean ± SD. **P < 0.01; ***P < 0.001; ns, not significant using one-way ANOVA. (D) The cytotoxicity of the indicated doses of PTC596 against primary MM cells analyzed by Annexin V staining using flow cytometry. Primary MM cells were treated for 12–24 h. Data represent mean ± SD of duplicate experiments. *P < 0.05; **P < 0.01; ***P < 0.001 using Student’s t-test or one-way ANOVA.