HeLa cells were treated with indicated compounds at their respective EC90 for 20 hours, fixed with 4% parafor-maldehyde, permeabilized with 0.2% Triton X- 100/ PBS and co-stained for DNA (0.5 ug/ ml Hoechst 33342) and tubulin (rat anti-tubulin primary antibodies and anti-rat Cy3 secondary antibodies).

Consistently, a similar SAR trend was observed by evaluating each compound’s potency (EC50) in HeLa cells with regards to their ability to arrest cells in G2/M-phase and induce cell death.

To test if tubulin was the primary target, we treated HeLa cells with compounds 6—12 and analyzed their effects by IF microscopy.

As expected, compounds 6—12 induced a microtubule depolymerization phenotype in HeLa cells (Figs 5G and 812).

(AF) Immunofluorescence of HeLa cells treated with control DMSO or indicated compounds (1—5) for 20 hours.

(A) For cell Viability assays, HeLa cells were treated with increasing concentrations (20-point titration 0—-100 uM) of indicated compounds (6—12) for 20 hours and the percentage of cells arrested in G2/M was quantified.

Immunofluorescence microscopy of HeLa cells treated with control DMSO, Taxol, col-chicine, or the indicated compounds (6—12) for 20 hours.