Latrunculin a

Luciferase assays were performed in MDA-MB-231 cells with the novel YAP1/TAZ-responsive reporter Hsa.CTGF-Lux. The −200/+27-CTGF promoter fragment was cloned in the pGL3-basic Luciferase Reporter Vector (Promega). Luciferase reporters (50 ng/cm²) were transfected together with CMV-β-gal (75 ng/cm²) to normalize for transfection efficiency. Each sample was transfected in duplicate and each experiment was repeated at least three times independently. NF2 is Addgene #19701. Latrunculin A was from Santa Cruz. The sequences of the siRNA used in this study are as follows (sense strand sequences are indicated): YAP1 1: GACAUCUUCUGGUCAGAGA dTdT; YAP1 2: CUGGUCAGAGAUACUUCUU dTdT; TAZ 1: ACGUUGACUUAGGAACUUU dTdT; TAZ 2: AGGUACUUCCUCAAUCACA dTdT; control: UUCUCCGAACGUGUCACGU dTdT. YAP1/TAZ siRNA 1 refers to the mix composed of oligos YAP1 1 and TAZ 1; YAP1/TAZ siRNA 2 refers to the mix composed of oligos YAP1 2 and TAZ 2.

Cell culture media was purchased from Invitrogen. Pertussis toxin (PTX) and AMD3100 were from Sigma Aldrich, NSC23766 was from Tocris Bioscience, Latrunculin A and Jaspakinolide were from Santa Cruz Biotechnology, and recombinant CXCL12 from Peprotech. Cannulas were custom-made and purchased from Plastics One. pCMVR8.74 (Addgene plasmid #22036, RRID:Addgene_22036) and pMD2.G (Addgene plasmid #12259, RRID:Addgene_12259) were gifts from Didier Trono.

Yeast media were prepared as described in Adams et al. (1997) . Gene disruptions were performed as detailed in Sikorski and Hieter (1989) (link) and Taxis and Knop (2006) (link). Strains were cultured at 30° except where otherwise indicated. The genotypes of strains used in the course of this study, along with construction details, are provided in Supporting Information, Table S1. Oligonucleotides used during this study are listed in Table S2. Ethidium bromide (Thermo-Fisher Scientific, Waltham, MA) was used at a concentration of 25 µg/mL to destroy mtDNA. Cycloheximide (CHX; Sigma-Aldrich, St. Louis, MO) was used at a concentration of 10 µg/mL for plasmid counterselection on yeast extract peptone dextrose broth (YEPD) medium, 3 µg/mL for plasmid counterselection on yeast extract peptone 3% glycerol+3% ethanol (YEPGE) medium, and at 0.2 µg/mL in YEPD medium to test for activation of the PDR pathway. Ketoconazole (Tokyo Chemical Industry Co., Tokyo, Japan), another PDR substrate, was used at a concentration of 2 µg/mL in YEPD. To disrupt the actin cytoskeleton in logarithmic-phase cultures, latrunculin A (Santa Cruz Biotechnology, Dallas, TX) was used at a concentration of 10 µM in SD medium lacking leucine (SD-Leu), following the procedure of Hammermeister et al. (2010) (link). Serial dilution assays were performed as in Garipler et al. (2014) (link).