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This work was funded by a Cancer Research UK Programme Foundation Award to C

This work was funded by a Cancer Research UK Programme Foundation Award to C.B. translocate to the nucleus upon their activation by mechanical cuesin particular cell contractility and spreading16. The antibody used in these assays recognises both YAP and TAZ proteins. Nuclear translocation of YAP/TAZ serves as a proxy for activation of transcriptional activity17. Thus, by simultaneously quantifying the translocation of YAP/TAZ with cell shape, we can determine if gene depletion has affected cell shape in a manner Y-26763 that has resulted in the activation of YAP/TAZ mechanotransduction (i.e., increased spreading or contractility), or Smo whether the cell shape change has occurred through alternate means (i.e., decreased viability); which provides some mechanistic insight into how gene depletion may be affecting shape. This data will be useful for elucidating a Y-26763 group of RhoGEFs and RhoGAPs which contribute to the cell shape of TNBC cells and could drive TNBC metastasis. The identification of these genes would warrant their further study in 3D cultures, and finally (Dharmacon). Both libraries were made up of SMARTpools, where each gene is usually targeted by a pool of 4 different siRNA strands per siRNA. Each siRNA Y-26763 library was arrayed across 2 black, optically clear bottom, 384 well Cell Carrier plates (PerkinElmer). Individual siRNAs were arrayed in duplicate per plate, and each plate was screened in duplicate, resulting in 4 plates per screen per cell line: 1A, 1B, 2A and 2B. As positive controls (Dharmacon cat # M-004632-00) and (Dharmacon cat # L-012200-00) siRNA were introduced on columns 23 and 24 with a minimum of 4 wells per control per screen. siRNA was used as a control for our ability to assess YAP/TAZ localisation, as in the absence of LATS1 YAP/TAZ nuclear localisation is usually expected to increase14. siRNA was used as a control to assess our ability to monitor both YAP levels and localisation. As a control to assess our ability to transfect cells we used siRNA (Dharmacon cat # L-006450-00, # M-006450-00) which results in multinucleate cells23,24. YAP/TAZ nuclear translocation can be sensitive to cell density, and thus some siRNAs might affect YAP/TAZ localisation simply because they reduce cell numbers17. To account for density dependent effects cells were plated at increasing densities on columns 1, 2, 23 and 24. By performing a linear regression, or comparable analysis on these samples users can identify the relationship between YAP/TAZ localisation and cell density. However, in these screens presented here no siRNAs had significant effects on cell number. Mock transfected cells served as our unfavorable controls, due to our previous observation that non-targeting siRNAs result in phenotypic changes (unpublished data). All liquid dispensing steps were carried out using a Multidrop Dispenser (Thermo Scientific), except for siRNA plating around the assay plates which was carried out using the acoustic liquid handler Echo 550 (Labcyte Inc). Screening reagents Dharmacon siGENOME siRNA library. Dharmacon ONTARGETsiRNA library. Opti-MEM Reduced Serum Media (Thermo Fisher, Gibco Life Technologies, cat # 31985062). Lipofectamine ? RNAimax reagent (Invitrogen, cat # 13778150). 8% methanol free formaldehyde in PBS (16% pre-made solution Thermo Scientific, cat # 28908), final concentration 4%. PBS made up of 0.05% of the preservative Sodium Azide (Sigma, cat # S2002-25G) (all PBS referred to in this study). Triton-X-100 (Sigma, cat # T9284), 10% in PBS. Bovine Serum Albumin (BSA) (Sigma, cat # A2153-50G) 2% in Y-26763 PBS, filtered. Triton-X-100, 0.01% and 0.5% BSA in PBS solution for antibody incubations. Mouse YAP/TAZ antibody (Santa Cruz Biotechnology, cat # sc-101199) 1?gml?1 (1:1,000). Rat Y-26763 -tubulin antibody (Bio-Rad, cat # MCA77G) 1?gml?1 (1:1,000). AlexaFluor 647 goat anti-mouse (Life Technologies, cat # A21235) 2?gml?1 (1:1,000). AlexaFluor 568 goat anti-rat 1:1,000 (Life Technologies, cat # A11077) 2?gml?1 (1:1,000). Phalloidin 488 (Invitrogen, cat # A12379) 1:1,000 from 300?U in 1.5?ml stock. 2-(4-Hydroxyphenyl)-5-(4-methyl-1-piperazinyl)-2,5-bi(1H-benzimidazole) trihydrochloride (Hoechst) (Sigma Aldrich, cat # 33258) 10?gml?1 (1:1,000). Day 0: Plating siRNAs 40?nl (0.08?pmol) siRNA from the siGENOME and ONTARGETlibraries (stock concentration of 20?M) were arrayed using the.