FIGURE 5:

Paclitaxel stabilization of microtubules increases Salmonella-induced pyroptosis and cell death due to a pore-forming toxin. (A) Paclitaxel and colchicine both increase Salmonella-induced pyroptosis. THP-1s were treated for 30 min with 500 nM paclitaxel or colchicine before infection with S. typhimurium MOI30. Cells were assayed with 7-AAD staining and flow cytometry at 3 h after initial infection. (B) Paclitaxel increases pyroptosis as measured by release of cytosolic protein. THP-1s were treated for 30 min with 500 nM paclitaxel before infection with S. typhimurium MOI30. Cells were assayed with Cytotox-Glo at 3 h after initial infection. (C) Paclitaxel does not alter active IL-1β release from THP-1s presimulated overnight with LPS (20 ng/ml) and then infected with S. typhimurium (MOI30, 3 h), whereas colchicine results in a substantial increase. (D) Paclitaxel increases F. novicida–induced pyroptosis. THP-1s were incubated overnight with 20 ng/ml LPS and treated with paclitaxel (500 nM) for 30 min before infection. (E) Paclitaxel and colchicine dose–response curves for polymerized microtubule mass and pyroptosis indicate that microtubule stabilization progressively increases pyroptosis. Paclitaxel or colchicine was added to cells 30 min before infection at the indicated doses. Colchicine had no effect on pyroptosis except at the highest dose of colchicine (500 nM), which resulted in near-complete loss of microtubules. Paclitaxel gradually increased polymerized microtubule mass and also caused a gradual increase in pyroptosis until 100 nM, at which further stabilization of microtubules had no further effect on pyroptosis. Inset micrographs are of microtubule staining at 0 and 500 nM paclitaxel and 500 nM colchicine. (F) Microtubules regulate cell death induced by the pore-forming toxin streptolysin O. THP-1s were treated for 30 min with 500 nM paclitaxel, 500 nM colchicine, or buffer before addition of streptolysin O (200 U). Cells were assayed with 7-AAD staining and flow cytometry at 90 min and 3 h after addition of streptolysin O. Paclitaxel increased cell death, whereas colchicine decreased cell death. (G) Microtubules have no effect on cell death induced by the detergent Triton X-100. THP-1s were treated for 30 min with 500 nM paclitaxel, 500 nM colchicine, or buffer before addition of Triton X-100 (250 μM). Cells were assayed with 7-AAD staining and flow cytometry at 45 min, 90 min, and 3 h after addition of Triton X-100. No effect on cell death was seen with either paclitaxel or colchicine. For A–F data are mean ± SEM for three or four separate experiments, and p value is for paired t test.