Figure 5.

SP600125 increases G₂/M arrest and endoreduplication through tubulin polymerization. (A) Tubulin polymerization was analyzed using immunofluorescent staining in U937 cells treated with 20 µM SP600125 for the indicated times and with 5 nM nocodazole (Noc) or 5 nM paclitaxel (Tax) at 1 h. Cells were fixed, permeabilized, and stained with anti-α-tubulin monoclonal antibody. Monoclonal antibody was detected using an anti-mouse secondary antibody conjugated with Texas Red. Cells were analyzed using fluorescence microscopy (× 400). DAPI was used for nuclear staining. (B) Polymeric tubulin and monomeric tubulin were extracted from U937 leukemia cells treated with SP600125 (20 and 40 µM) for 48 h. Polymerized tubulin was also extracted from cells treated for 48 h with either 5 nM Noc or with 5 nM Tax to indicate the range from less than 5% polymeric tubulin to greater than 95% polymerized tubulin. The quantification of polymeric tubulin from three independent experiments is shown. (C) Effects of SP600125 on in vitro tubulin polymerization. MAP-rich tubulin (1 mg/ml) was incubated at 37℃ for 0-30 min, then treated with SP600125 (40 µM and 200 µM), 3 µM Noc, and 3 µM Tax. The indicated compounds were then added to the indicated concentrations. A340 values were recorded once every 3 min. The results are from one representative experiment of three performed that showed similar patterns.