25×104 in a final volume of 50 μL. A total of 721.221 target cells were added (1×104 in 50 μL) to each well (quadruplicate wells were assayed per point) and the plate was centrifuged at 500 rpm for 1 min and incubated for 4 h at 37°C. At the end of this incubation period, 50 μL of assay buffer was added to each well. The substrate (50 μL/well) was added and the samples incubated in dark for 15 min. The plates were read using Synergy4 microplate reader (BioTeK® Instruments). Maximum cell lysis was determined by treating 1×104 target cells with 0.1% digitonin in assay buffer for 3 min at RT. Freshly harvested YTS, control vector-transduced YTS, and IQGAP1 shRNA-transduced YTS cells washed and resuspended
in 0.5% BSA in PBS (PBS-BSA). The cells were fixed for 10 min FK506 ic50 at room temperature in PBS containing 2% paraformaldehyde, washed three times in BYL719 chemical structure PBS-BSA, and permeabilized
with 0.1% Tween-20 in PBS-BSA for 5 min. The cells were washed three times in PBS-BSA and incubated with primary Ab against IQGAP1 or Alexa fluor phalloidin 488 for 45 min. The cells were washed and incubated with secondary goat anti-rabbit Alexa fluor 488 for 45 min. Cells were washed and staining was assessed (10 000 cells/sample) using a BD FACS Array system. First, the live cells were gated to exclude debris, and then the number of cells positive for Alexa fluor 488 was assessed within this population. The assay was performed according to the method described in 26. YTS cells were prelabeled with 1.5 μM Cell Tracker™ Green CMFDA (Invitrogen cat no. C2925) and target cells were labeled with 5 μM Cell Tracker™ Orange (Invitrogen cat no. C34551). The PDK4 cells were combined
at an effector to target ratio of 2:1 and incubated for the indicated times. The samples were gently vortexed for 3 s at maximum vortex speed and immediately fixed with 2% PFA. Samples were run in triplicates and 30 000 events were counted for every replicate. The frequency of double-positive events was determined within the Cell Tracker™ Green-positive population using Summit V188.8.131.5277 software. The following gating strategy was used: First, the live cells were gated to exclude debris. Compensation adjustments were made on this population using single-positive cells stained for either of the two dyes. Gates were set to differentiate between the double positives, represented in G2, from the single positives and double negatives in the experimental cells. This research was supported by a grant from the Canada Institutes for Health Research (J. A. W.) and the Health Sciences Research Department (N. K.). The authors thank Qiujang Du for preparation of the shRNA-mir constructs and Monroe Chan for flow cytometry. Conflict of interest: The authors declare no financial or commercial conflict of interest. Detailed facts of importance to specialist readers are published as ”Supporting Information”.