Black walled plates

The stably transfected RKO (pBARLRen) cells were used as described for the chemical compound library screen. Cells were plated at ~2400 per well of 384-well black-walled plates (Greiner) pre-spotted with 10 nanomoles of either individual siRNAs (ON-TARGETplus, Dharmacon/GE Healthcare) or siRNA inert sequence control (Dharmacon) listed in Supplemental Table 1 in Lipofectamine RNAiMAX (ThermoFisher). Cells were cultured for 2 days to allow siRNA-mediated knockdown of cognate protein expression. Recombinant Wnt3a (10% final concentration of Wnt3a conditioned medium, as above) together with either PAWI-2 (100 nM) or DMSO vehicle was added to each well and luciferase activity was determined 6 hours later using SteadyLite HTS (PerkinElmer). PAWI-2 and DMSO vehicle conditions for each siRNA were tested in quadruplicate wells. PAWI-2 activity was normalized relative to the corresponding DMSO control. The effect of each siRNA on PAWI-2 activity was then calculated as % inhibition relative to that observed in the control siRNA samples (0% inhibition). These activities were then expressed as a Z-score to account for variation between plates. Most siRNAs were ± 1 standard deviation from the control siRNA, and hence considered to have negligible activity.

IMA cells were plated at 1000 cells per well in 6 well plates for long term growth assays or 2000 cells per well in 96 well plates for short term growth assays. For long term growth assays, plates were fixed in methanol and stained with 0.2% crystal violet solution before imaging plates on a Chemi-doc imager (Bio-Rad). Areas were determined in ImageJ using the Colony Area plugin. For short term growth assays cells were plated in 96-well black walled plates (Greiner), cultured for 6 days, and then processed following the manufacturer’s recommended protocol for the Cyquant assay (Invitrogen).