Figure 5

AUY922 does not affect microtubule polymerization in prostate cancer cells. (A) AUY922, 17-AAG and control compounds at the indicated doses were incubated with purified tubulin protein at 37 °C, and polymerization was monitored using a FLUOstar Omega plate reader at 340 nm every 1 min for 60 min at 37 °C. (B) Representative images of LNCaP cells treated with AUY922 (31 or 250 nM), nocodazole (NOC) (250 nM) or vehicle control (DMSO) for 48 h and subjected to fluorescence microscopy of α-tubulin (green) and DNA (blue) using an INCell 2200 automated imaging system at 40x magnification. Cells treated with DMSO or AUY922 showed intact microtubule network, while cells treated with the microtubule destabilizing drug NOC displayed depolymerized microtubules. Scale bar = 5 µm. (C) AUY922 (15–500 nM) did not affect α-tubulin polymer mass, while NOC (250 nM) and paclitaxel (200 nM) reduced and increased, respectively, α-tubulin polymer mass, as shown by quantitative immunofluorescence microscopy of the cellular α-tubulin mean intensity as depicted in (B) (expressed as fold-change relative to DMSO control, n = 3, mean ± SD).