Supplementary Note 1. Materials and Methods for the BRET procedure. Solutions and reagents

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1 Supplementary Note 1 Materials and Methods for the BRET procedure Solutions and reagents PEI (1 mg/ml): The polyethyleneimine (PEI) powder (Polysciences, Inc.; MW ~25 KDa; cat#: 23966) is dissolved in bi-distilled water (ph adjusted with 1N HCl added dropwise until most insoluble materials disappear). The stock solution is then filtersterilized, aliquoted (5 ml) and stored at -20 C. Coelenterazine 400a (1 mm): Coelenterazine 400a was purchased from either Biotium (cat#: ) or Gold Biotechnology (cat#: C ). Both sources produced similar results. Stock solutions are prepared in 100% anhydrous ethanol and stored at -20 C as 1ml aliquots. For increased longevity, aliquots are stored in the presence of a desiccant. NOTE: Coelenterazine 400a precipitates in aqueous solution within ~ 30 minutes and tends to flocculate during HTS runs. This is circumvented by adding 1% Pluronic F-127 to the Coelenterazine 400a solution. Pluronic F-127 (10%): The (weight/volume) solution is prepared in water and filtersterilized. Gentle warming accelerates dissolution. The powder was purchased from Sigma (cat#: P2443). 10X Tyrode s solution without CaCl 2 : 250 mm Hepes (ph 7.4), 1.4 M NaCl, 27 mm KCl, 120 mm NaHCO 3, 56 mm D-glucose, 4.9 mm MgCl 2, 3.7 mm NaH 2 PO 4. The 10X stock solution is prepared without CaCl 2 (CaCl 2 precipitates with phosphate otherwise). Adjust ph with NaOH. The 1X Tyrode s solution also contains 1 mm CaCl 2. Plates White opaque 96-well format plates (BD Biosciences, cat#:353296) were used for lowthroughput assays or 384-well format plates (Greiner Bio One, cat#: ) for highthroughput screening. Cells HEK293T cells were used for all BRET assays and cultured in complete DMEM medium, which includes high-glucose + pyruvate + L-glutamine (Invitrogen, cat#: ) supplemented with 10% heat inactivated fetal bovine serum (FBS, HyClone) and 1% Pen/Strep (Invitrogen). Cells were first amplified in batch and frozen (25X10 6 per 1ml aliquots) to obtain a homogenous pool of cells at a similar passage number. 1

2 Procedure 1: BRET titration curves This protocol describes the procedure for conducting BRET titration curves following transient transfection. Day 0: Cell plating One day prior to transfection, HEK293T cells are seeded in 6-well plates (BD Biosciences, cat#:353224; 3X10 5 cells per well). Day 1: Cell transfection Fixed amounts of RlucII constructs (50 ng in the example) and a varying amount of GFP10 constructs (0 to 1000 ng in the example) are transfected using PEI. Salmon sperm DNA (ssdna) is used to equalize total amount of transfected DNA between wells. 1- DNA dilutions: Three separate columns of a 96-well plate are populated with: Column #1 - RlucII constructs: A single dilution of RlucII donor construct (5 ng/ l) is dispensed in each well of Column #1 of a 96-well microtiter plate. Column #2 - GFP10 constructs: Two-fold serial dilutions of the GFP10 constructs are then prepared in Column #2. No DNA is added to Row H and will serve as the BRET blank (luciferase alone transfection). Column #3 - salmon sperm DNA: Varying concentration of salmon sperm DNA (ssdna) is used to complement the DNA present in each well. The various dilutions are prepared to obtain a total of 1050 ng of DNA in each well of the transfection plate. The wells of Row A contain no ssdna, while Row H contains a 100 ng/ul dilution. RlucII constructs (ng/ l) GFP10 constructs (ng/ l) ssdna (ng/ l) A B C D E F G H NOTE: DNA dilutions in sealed plates can be stored at -20 C for at least 2 months. 2- Transfection plates Transfections for BRET titration experiments are systematically conducted in triplicate. This represents 24 transfections for a single titration experiment. i. Transfer 10 l of the RlucII dilution to three columns of a new 96-well plate (each transfection is done in triplicate). ii. Transfer 10 l of the GFP10 constructs of varying concentrations to wells containing RlucII constructs. 2

3 iii. Transfer 10 l of ssdna of varying concentrations to the appropriate wells. iv. Add 100 l of plain DMEM medium to each well. v. In a separate plate, mix 6 l of PEI (1 g/ l) l of plain DMEM medium. vi. Dispense 100 l of the PEI/DMEM mixture to each well of the transfection plate. Mix up and down. Incubate 15 min. at room temperature. In the mean time replace media from the 6-well HEK293T plates with fresh complete media. 3- Transfection Add the transfection mix (~330 l) to the cells and incubate for 48h at 37 C; 5% CO 2. Day 3: BRET readings i. Remove media by gentle aspiration. ii. Wash cells with 1ml of 1X Tyrode s solution. iii. Resuspend cells in 500 µl of 1X Tyrode s buffer by gentle up and down pipetting. iv. Dispense 90 µl of cell suspension to a white opaque microtiter plate (1 well of a 6-well plate is enough for five BRET readings). v. Read GFP10 fluorescence on a FlexStation II (Molecular Devices) with excitation and emission peaks set at 400 nm and 510 nm, respectively. vi. Add 10 µl of 25 M Coelenterazine 400a (1/40 dilution of the 1 mm stock solution) to each assay well (2.5 µm final concentration). vii. Incubate cells 10 min., then read BRET signals on a Mithras LB940 plate reader (Berthold Technologies) equipped with donor/acceptor emission filters (donor 480 ± 20 nm/acceptor 530 ± 20 nm). 3

4 Procedure 2: BRET-based dose-response experiments This protocol describes the procedure for conducting dose-response experiments using BRET probes following transient transfection. The procedure is described for the RlucII- BRAF KD -CAAX / GFP10-BRAF KD -CAAX probes. Day 0: Cell plating One day prior to transfection, HEK293T cells are seeded in 100 mm dishes (BD Biosciences, cat#:353003; 2.5X10 6 cells per dish). Day 1: Cell transfection i. Prepare two tubes that will contain Mix1 (DNA dilution) and Mix2 (PEI dilution). To transfect a single 100 mm petri dish, the following amounts of reagents are used: Mix1: 800 l of complete DMEM 0.8 l (0.4 g) of RlucII-BRAF KD -CAAX (0.5 g/ l) 4 l (2 g) of GFP10-BRAF KD -CAAX (0.5 g/ l) - Vortex thoroughly Mix2: 800 l of complete DMEM 48 l of PEI (1 g/μl) - Vortex thoroughly ii. Mix together the content of Mix1 and Mix2 and vortex again. iii. Incubate for 15 min. at room temperature. iv. Dilute the transfection mix in 10 ml of complete DMEM medium. v. Remove cell culture media from the cells. vi. Slowly add the transfection mix + medium to the cells (on the side of the dish) and incubate for 48h at 37 C; 5% CO 2. Day 3: Cell treatment with compounds and BRET readings 1- Cell preparation i. Remove cell media by gentle aspiration. ii. Gently wash cells with 4 ml of 1X PBS. iii. Add 1ml of Trypsin-EDTA solution (Invitrogen, cat#: ) and incubate for 1 min. at 37 C or until the cells have detached from the dish. iv. Add 9 ml of complete DMEM medium to inactivate trypsin and dissociate cells by gentle up-and-down pipetting. v. Spin the cells at 250 rpm for 5 min. at room temperature. vi. Remove supernatant and wash cells with 5 ml of 1X Tyrode s buffer. vii. Spin cells at 250 rpm for 5 min. at room temperature. 4

5 viii. Remove supernatant and resuspend the cells in 4 ml of 1X Tyrode s solution. ix. Dilute cells at 1X10 6 cells/ml. x. Dispense cell suspension (90 l/well) in 96-well white opaque microtiter plates (BD Biosciences, cat#:353296). NOTE: A single 100 mm plate typically yields enough cells to perform dose-response assays in a full 96-well plate. 2- Compound dilutions Compound dilutions were prepared in DMSO to obtain 10 concentrations ranging from 0 (DMSO alone) to 10 mm. These DMSO dilutions can be kept for several months at -80 depending on compounds stability. 10,000 3,333 1, DMSO dilution concentration (μm) The DMSO dilutions were further diluted 1:100 in a final volume of 200 l of 1X Tyrode s buffer: 100,000 33,333 10,000 3,333 1, Intermediate dilution concentration (nm) 3- Cell treatment and BRET readings 10 l of each 1:100 compound intermediate dilution is added to cells in the 96-well assay plate. 10,000 3,333 1, Final concentration on cells (nm) i. Incubate cells for 1h at 37 C; 5% CO 2 upon compound treatment. ii. During this 1h incubation, read intrinsic GFP10 fluorescence signals on a FlexStation II reader (Molecular Devices) with excitation and emission peaks set at 400 and 510 nm, respectively. iii. 10 minutes prior the end of the 1h incubation, add 10 l of 25 M Coelenterazine 400a (1/40 dilution of the 1 mm stock solution) to each well (2.5 M final concentration). iv. Read BRET signals on a Mithras LB940 plate reader (Berthold Technologies) equipped with donor/acceptor emission filters (donor 480 ± 20 nm/acceptor 530 ± 20 nm). NOTE: The 10 min. incubation time with Coelenterazine 400a and the time required to add the substrate is included within 1h incubation time with the compounds. 5

6 Procedure 3: high-throughput screening with RAF/RAF BRET biosensors This protocol describes the procedure for conducting a high-throughput chemical screen using BRET probes following transient transfection. The procedure is described for the RlucII-CRAF KD -CAAX / GFP10-BRAF KD -CAAX probes. Note: Volumes and quantities described are for ~ 20 assay plates (384-well format). Day 0: Cell plating i. Thaw one (1) vial of HEK293T cells (25X10 6 ) for 1 min. at 37 C. ii. Wash cells once in 50 ml of sterile PBS and pellet cells at 250 rpm for 5 minutes at room temperature. iii. Resuspend cells in complete DMEM medium and seed 16.6X10 6 cells in 250 ml of complete medium per Double CellStack (Corning) or 12.5X10 6 cells in 200 ml of complete medium per HiFlask (Millipore). Grow cells for three days at 37 C; 5% CO 2. Day 3: Cell transfection i. Prepare two tubes that will contain Mix1 (DNA dilution) and Mix2 (PEI dilution). To transfect a double CellStack or one HiFlask, the following amounts of reagents are used: Mix1: 12.6 ml of complete DMEM l of RlucII-CRAF KD -CAAX (0.5 g/ l) l of GFP10-BRAF KD -CAAX (0.5 g/ l) - Vortex thoroughly Mix2: 12.6 ml of complete DMEM l of PEI (1 g/μl) - Vortex thoroughly ii. Mix together the content of Mix1 and Mix2 and vortex again. iii. Incubate for 15 min. at room temperature. iv. Dilute the transfection mix in a final volume of 250 ml (for a Double CellStack) or 200 ml (for a HiFlask) of complete DMEM. v. Slowly add the transfection mix + medium to the cells and incubate for 48h at 37 C; 5% CO2. 6

7 Day 5: HTS run 1. Cell harvesting and dispensing i. Wash cells with 50 ml of PBS (thoroughly wash of each layer of the CellStack or HiFlask). Decant PBS in a beaker. ii. Add 15 ml of Trypsin (0.05%) per CellStack or HiFlask. iii. Incubate cells at 37 C for 1 to 5 minutes until complete dissociation. NOTE: This is important because cell clumps affect reproducibility of screening outputs. Avoid over trypsinization which causes significant cell lysis. iv. Inactivate trypsin with 35 ml of complete DMEM and transfer cells to two 50 ml Falcon tubes (~25 ml/tube). NOTE: Thoroughly and rapidly mix trypsin and complete medium on each layer of the cell culture vessel to minimize cell lysis. v. Rinse each CellStack or HiFlask with an additional 50 ml of complete medium to maximize cell harvest. Transfer this rinsing volume to each of the two Falcon tubes from the previous step (~25ml/tube). vi. Spin cells 250 rpm for 5 min. at room temperature. vii. Discard the supernatant and wash cell pellet with 20 ml of 1X Tyrode s buffer for each Falcon tube. viii. Spin cells 250 rpm for 5 min. at room temperature. ix. Discard the supernatant and pool the contents of the two Falcon tubes in 200 ml of 1X Tyrode s solution. x. Count cells with a hemacytometer and dilute at 1.33X10 6 cells/ml in 1X Tyrode s solution. xi. Dispense cell suspension (45 l/well; 6X10 4 cells) in 384-well assay plates using a Multidrop Combi. 2. HTS assay General comments Cells are maintained at 37 C in a CytoMat automated incubator during the entire course of the procedure except when adding compounds, Coelenterazine 400a or when reading intrinsic GFP10 fluorescence (on an EnVision plate reader) and the BRET signals (on a Spectramax L plate reader). Cells are treated for a total of 2 hours with library compounds prior to addition of Coelenterazine 400a. 7

8 The library is kept in V-bottom 384-well plates. Rows 1, 2, 23 and 24 contain DMSO alone and are used as negative controls. The layout of compound plates is provided in Figure A. Figure A. Compound plate layout DMSO Compounds Four control rows were present in each assay plate: rows 1 and 23 contained DMSO alone (negative controls), whereas rows 2 and 24 contained GDC-0879 and served as positive controls for dimerization induction (Figure B). These rows were used to calculate the Z-factor for each plate during the screen. Figure B. Assay plate layout GDC-0879 (100nM) DMSO Compounds 8

9 Solutions required for a HTS run Before starting the run, appropriate volumes of the following solutions are freshly prepared: 1 M GDC-0879 in 1X Tyrode s solution from a 10 mm stock solution (0.01% DMSO residual). 0.01% DMSO in 1X Tyrode s solution. 25 M Coelenterazine 400a; 1% pluronic F-127 in 1X Tyrode s solution. Robotic Procedure The robotic procedure for the screen was as follows: i. Bring assay and compound plates to the Biomek workspace by the robotic arm. ii. Add 0.25 l of compound (2 mm) per assay well using a 384-tips pin tool to obtain a final concentration of 10 M. NOTE: final DMSO concentration in assay wells is 0.5%. NOTE: the pin tool is calibrated with FITC diluted in DMSO. iii. Add 5 l of 0.01 % DMSO to control rows 1 and 23 (to reach the same DMSO concentration as for GDC-0879-treated wells; 0.001% DMSO). iv. Send back assay and compound plates to initial positions (i.e., CytoMat and Carrousel, respectively). v. Incubate assay plates for 1h in CytoMat. vi. Bring assay plates to the Multidrop primed with 1 M GDC-0879 in 1X Tyrode s solution and add 5μl to rows 2 and 24. vii. Send back assay plates to Cytomat and incubate for 1h. viii. Bring assay plates to the EnVision and read GFP10 signals using a 400 nm excitation filter and a 510 nm emission filter. ix. Send assay plates to the Multidrop, add 5 l of Coelenterazine 400a for a final concentration of 2.5 M and send back to Cytomat for 10 min. incubation. x. Read BRET signals on a SpectraMax L plate reader equipped with 2 PMTs with a BRET_dual_read protocol using a 480 nm luciferase emission filter and a 535 nm GFP10 emission filter. NOTE: BRET signals emitted by RlucII / GFP10 pairs (BRET2) can be read with either BRET1 or BRET2 filter sets. The main difference is that the calculated BRET ratio is higher with BRET1 filters (480 nm and 530 nm) than with BRET2 filters (400 and 510) since only the shoulder of the RlucII emission spectrum is captured, while the full peak is detected with the 400 nm filter. BRET1 filters produce slightly better Z-factors. HTS BRET assays are run on a platform comprising a Biomek liquid handler, and multiple instruments all coordinated with the SAMI software. A typical run lasts approximately 17h. 9