On targetplus non targeting pool d 001810 10

MCF7, MDA-MB-231, and Rat2 cells were maintained in Dulbecco Modified Eagle’s Medium (DMEM) and BT549 in RPMI 1640 medium. All media contained 10% fetal bovine serum (FBS, Invitrogen) and 1% penicillin-streptomycin. Recombinant Wnt5a protein (R&D, #645WN) and Wnt3a (Peprotech, #315–20) were used at 200 ng/mL. Actinomycin D (Sigma-Aldrich A-9415) was added at 40 ng/mL or 1000 ng/mL MCF7 and BT549 cells expressing exogenous Wnt5a were generated by infection with the retrovirus vector LNCX containing Wnt5a cDNA, followed by selection of pooled colonies in G418 (Geneticin). Control cells were infected in parallel with the ‘empty’ vector. Rat2 fibroblasts transduced with FLAG-tagged DVL1 in an LNCX vector were constructed by Dr. José González-Sancho. Lentivirus particles expressing DVL1 shRNA (sc-35228-V), or non-silencing control shRNA were purchased from Santa Cruz Biotechnology Inc. and used to infect MCF7 and BT549 cells with selection in 5 μg/mL puromycin. ON-TARGETplus Human DVL1 (1855) siRNA—SMARTpool (L-004068-00-0005) and ON-TARGETplus Non-Targeting Pool (D-001810-10 from Dharmacon was used for nucleofection (Lonza Group Ltd, Switzerland) of MCF7 cells.

Three β-catenin siRNAs (ON-TARGETplus SMARTpool siRNA L-003482-00, #9, #10, and #12) and control siRNA (ON-TARGETplus Non-targeting Pool #D-001810-10) were purchased from GE Dharmacon (Lafayette, CO). Target sequences of the siRNAs are shown in Supplementary Table 2. HCT116 or SW480 were seeded a day before the treatment with siRNA, and transfected with 15 nM of β-catenin or control siRNA using Lipofectamine RNAiMAX (Thermo Fisher Scientific). Forty-eight hours after the transfection, RNA and proteins were extracted from the cells. The silencing effect of β-catenin siRNAs was evaluated by quantitative RT-PCR and western blotting.

All siRNA transfections were performed using Lipofectamine RNAi MAX (Invitrogen) following the manufacturer's recommendations. The final concentration of the siRNA molecules is 10 nM and cells were collected 72 or 96 h later according to the purposes of the experiments. Control siRNA (ON-TARGETplus Non-Targeting Pool, D-001810-10), USP9X siRNA (ON-TARGETplus, L-006099-00-0005) and CEP131 siRNA (ON-TARGETplus, L-023335-00-0005) were from Dharmacon in a smart pool manner, while the individual siRNAs against USP9X (USP9X siRNA-1: 5′-GTCGTTACAGCTAGTATTT-3′, USP9X siRNA-2: 5′-CTGTGATTCAGCAACTCTA-3′, USP9X siRNA-3/3′UTR: 5′-GAGAGTTTATTCACTGTCTTA-3′), PCM1 (PCM1 siRNA-1: 5′-CCAATGATATTTCTCCGGA-3′, PCM1 siRNA-2: 5′-CAGACTTCCCTCCAGGCTA-3′), CDK2 (5′-GAGCUUAACCAUCCUAAUA-3′), MCL1 (5′-GAAATTCTTTCACTTCATT-3′), ITCH (5′-ACATGCCATCTACCGTCATTA-3′) and USP33 (5′-GAUCAUGUGGCGAAGCAUA-3′) were chemically synthesized by Sigma (Shanghai, China). The short hairpin RNAs (shRNAs) against USP9X and CEP131 in pLKO vectors were purchased from Sigma.