Figure 7.

Paclitaxel did not influence macrophage priming in the process of NLRP3 inflammasome activation. (A) BMDMs were treated with LPS (500 ng/ml), paclitaxel (100 nM) and epothilone B (100 nM) for 4 h. Indicated proteins in the cell lysates were analyzed by Western blotting. Actin was used as a loading control. (B–D) LPS-primed BMDMs were pre-treated with graded doses of epothilone B for 1 h, followed by incubation with ATP (2 mM) for 30 min. (B,C) Cells were stained by Hoechst 33342 (blue; for all cells) and propidium iodide (PI) (red; for dead cells) for 10 min. (B) All images were captured by fluorescence microscopy, and the merged images show PI and Hoechst 33342 fluorescence with bright-field images. One set of representative images of three independent experiments are shown. Scale bars, 50 μm. (C) PI-positive cells in 5 randomly chosen fields (one field per well) each containing ~100 cells were quantified. The percentage of cell death is defined as the ratio of PI-positive relative to all (revealed by Hoechst 33342) cells. (D) The levels of soluble IL-1β in culture supernatants were analyzed by cytometric bead array (CBA) assay. (C,D) Data were analyzed using the one-way ANOVA followed by Turkey post-hoc test, which are shown as mean ± SD (n = 5). ^*P < 0.05; ^**P < 0.01; ^***P < 0.001; PTX, paclitaxel; EpoB, Epothilone B.