Evaluation of 3,5-Diphenyl-2-Pyrazolines for Antimitotic Activity by Inhibition of Tubulin Polymerization

Figure 6

effect of derivative 4 on mitotic spindle formation. HCT116 cells cultured on coverslips were treated with 40 μM derivative 4 for 24 h The cells were fixed and incubated with antibodies against α,β-tubulin and pericentrin for 2 h followed by incubation with an AlexaFluor 488-conjugated (for α,β-tubulin, green signal) and AlexaFluor 555-conjugated (for pericentrin, red spot) secondary antibodies for 30 min. Nuclear DNA was stained with 1 μg/mL Hoechst 33258 for 10 min (blue signal). M phase cells were selected and analyzed for assembly of mitotic spindles. The fluorescent signals were imaged using an EVOSf1® fluorescence microscope. Arrows, interphase cells (int); arrowheads, metaphase spindle (meta); star-like, telophase spindle (telo). Scale bars, 50 μm.