Sirna molecules

HCT 116 cells were treated with siRNA molecules (Dharmacon) and used to perform the RNA-seq and the ChIP-seq (p53 antibody FL-393, Santa Cruz Biotechnology sc-6243) experiments. The sequencing data were analyzed with standard bioinformatics pipelines. p53crys (human p53 [UniProt accession code P04637] residues 62 to 292) and iASPPcrys (human iASPP [UniProt Q8WUF5] residues 625 to 828) were expressed in Escherichia coli and purified to homogeneity. The p53–iASPP complex was prepared as an equimolar mixture of the purified proteins before size exclusion chromatography. The peak fractions were combined and concentrated for crystallization trials. Diffraction-quality crystals were optimized in 18% (wt/vol) polyethylene glycol 3350, 0.18 M trisodium citrate. Crystallographic data were collected and processed, and the structure was determined and analyzed, essentially as described previously (73 (link)). A detailed description of the materials and methods used in this study is provided in SI Appendix, Supplementary Materials and Methods. Raw data files as noted in the legends of Figs. 2, 3, and 6 are available from Mendeley Data at http://dx.doi.org/10.17632/j75wt9b36n.1.

Conditions established for high-throughput siRNA transfection in a 96-well format were as follows: CFBE41o⁻ cells expressing F508del-CFTR and the HS-YFP were reverse transfected with 10 nM (final concentration) siRNAs using lipofectamine 2000 as transfection agent. Twenty-four hours after transfection and plating, the medium was changed and the cells were incubated at 37 °C for additional 24 hours, prior to proceeding with the functional HS-YFP-based assay.
Depending on availability, we utilized siRNA molecules from Dharmacon (pools containing three different duplexes per target) or from Sigma (four different duplexes per target). Results were confirmed using Stealth siRNA molecules from Life Technologies (three duplexes per target). Sequences and/or catalog numbers of siRNAs will be provided upon request. Target silencing was confirmed by evaluating expression of the target mRNA.

MDA-MB-231 and BrM were transfected with SMARTpool siRNA (Dharmacon) targeting CD146 alongside a control scrambled (Scr) siRNA. Transfections were performed using Lipofectamine 200 RNAiMax (Invitrogen) transfection agent and Opti-MEM I Reduced Serum Medium, GlutaMAX Supplement (Gibco) according to manufacturer’s instructions. Cells were transfected with 30 pmol siRNA in a six-well plate (2–4 × 10⁵ cells/well) and scaled accordingly. Briefly, for a single well of a six-well plate, 30 pmol siRNA duplexes were made in 250 μL of OptiMEM medium and incubated at room temperature for 5 min. At the same time, 5 μL Lipofectamine was made up in 250 μL of OptiMEM and incubated at room temperature for 5 min siRNA and Lipofectamine mix were combined within the six-well plate and gently mixed before incubating at room temperature for 20 min. Following this, OptiMEM suspended cells were added to siRNA Lipofectamine complexes at 2–4 × 10⁵ cells in 1 mL of OptiMEM. Cells were incubated in this mixture for 4–6 h, before transfection medium was aspirated and replaced with supplemented normal culture medium. siRNA-treated cells were incubated for 24–72 h before being used in downstream assays. The siRNA molecules used (from Dharmacon/Horizon Discovery) are shown in Supplementary Table S1.