Research Paper Volume 12, Issue 4 pp 3713—3729

Identification of a lathyrane-type diterpenoid EM-E-11-4 as a novel paclitaxel resistance reversing agent with multiple mechanisms of action

Figure 6. EM-E-11-4 enhanced the effect of paclitaxel-induced tubulin polymerization in vitro. (A) Tubulin polymerization assay. Purified porcine tubulin in reaction buffer was incubated at 37°C with GTP in the absence or presence of the indicated agents. Tubulin polymerization was measured using a fluorescence microplate reader (ex = 370 nm, em = 445 nm) every 1 min for 60min. (B) A549/Tax cells were treated with EM-E-11-4 (2.5, 5, and 10 μM) and/or 100 nM paclitaxel for 48 h. Then, the soluble tubulin and insoluble tubulin were isolated, and the levels of α-tubulin were determined the by Western blot analysis. (C) Hela/βIII cells were treated with EM-E-11-4 (2.5, 5, and 10 μM) and/or 100 nM paclitaxel for 48 h. Then, the soluble tubulin and insoluble tubulin were isolated, and the levels of α-tubulin were determined by Western blot analysis. Columns represent the means±SD values for protein levels obtained from three individual experiments. * p<0.05 and ** p<0.01 vs. Soluble control (A: paclitaxel 100 nM; B: paclitaxel 20 nM), # p<0.05 and ## p<0.01 vs. Insoluble control (A: paclitaxel 100 nM; B: paclitaxel 20 nM).