Combining TQ and the PAK inhibitor IPA-3 increases apoptosis. A. HCT116wt cells, pretreated with different concentrations of IPA-3 (1-50 μM) alone or with TQ 40 μM for 24 hours, were stained with crystal violet dye and assessed for viability. Data are presented as percentage of control. *indicates a p-value < 0.001 when comparing IPA-3 treatment to IPA-3 + TQ treatment. Each value is the mean ± SD of three independent experiments each done in quadruplicates. B. Cells pretreated with 10 μM IPA-3 for one hour followed by 24 hours incubation with 40 μM TQ were collected and analyzed by flow cytometry for a double AnnexinV/PI staining. The percentage of the different cell populations was calculated by FlowJo software. C. Cells were pretreated for 1 hour with 10 μM IPA-3 and then incubated with 40 μM TQ. Cell pellets were collected after 1, 3, 6, 24 and 48 hours. Lysates were blotted against PARP and GAPDH for loading control. D. PARP cleavage was detected in endogenous (left blots) and kinase-dead dominant negative PAK1 K299R mutant transfected HCT116 wt cells (right blots) after 24 hours of 100 μM TQ. Higher TQ concentration was used in all transfection experiments due to the high cell density. E. Cells were transfected with PAK1 siRNA or scrambled (SCR) siRNA and checked for PAK1 knockdown by western blotting. PARP cleavage was detected in endogenous and PAK1 siRNA transfected cells after 24 hours of 60 μM TQ. SCR siRNA was used as transfection control. GAPDH was used as loading control. F. Cell lysates were immunoblotted against pPAKThr212, pPAKThr423 and total PAK1. GAPDH was used as a loading control. Data shown are representative of two independent experiments.