Figure 3

FRAX1036 and docetaxel (DTX) combine to alter stathmin phosphorylation, induce the apoptotic marker cleaved PARP and increase kinetics of apoptosis. (A) MDA-MB-175 and HCC2911 cells were treated with DMSO, 5 μM FRAX1036, 0.2 μM docetaxel and a combination of 5 μM FRAX1036 and 0.2 μM docetaxel for 24 hours. Cell lysates were immunoblotted with apoptotic and PAK1 downstream markers. (B) MDA-MB-175 cells were treated with DMSO or 0.2 μM docetaxel for 48 hours after non-targeting control short interfering RNA (siRNA) or PAK1 siRNA transfection for 72 hours. Cell lysates were harvested and subjected to immunoblot analysis for apoptotic markers and microtubule regulators. The molecular weight of the lower band from the phospho-stathmin immunoblot corresponds to total stathmin. The efficacy of knockdown by PAK1 siRNA was 47% (lane 2) and 80% (lane 4) as determined by densitometry. (C) Kinetic apoptosis assay. HCC2911 cells were plated in 96-well plates and were untreated (control) or treated with DMSO, 2.5 μM FRAX1036, 0.2 μM docetaxel, or a combination of 2.5 μM FRAX1036 and 0.2 μM docetaxel. Apoptosis was assayed by counting the number of green caspase 3/7-positive objects at each time point (Essen Cell player kinetic caspase 3/7 assay). (D) Apoptotic index. The number of apoptotic cells was normalized to the total number of cells at the final time point in (C) to account for cell proliferation. (E,F) The same as (C,D) with MDA-MB-175 cells. The average and SEM of three replicates are shown and a t-test performed at the final time point and on the apoptotic index (*P < 0.03, **P < 0.003, ***P ≤ 0.0001).