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  • br METHOD DETAILS br Cloning br

    2020-07-28


    METHOD DETAILS
    Cloning
    The human kinase library plasmid kit, containing open reading frames (ORFs) in Gateway Entry vectors, was provided by William Hahn and David Root (Johannessen et al., 2010; Yang et al., 2011) via Addgene (Kit # 1000000014). The human phosphatase library was obtained from Dharmacon (OHS4941, ORFeome Human Entry Collection Phosphatase). Destination vectors, including pDEST pcDNA5 FRT TO-eGFP and pDEST 30 Triple Flag pcDNA5 FRT TO, were kindly provided by Dr. Anne-Claude Gingras at Mount Sinai Hospital, Toronto, Canada (Couzens et al., 2013). Expression vectors encoding the FLAG- or GFP-tagged fusion proteins were generated via Gateway Cloning and sequenced before transfection. Vectors for kinase-dead mutants, including pFLAG-CMV-hErk1 (K71R) (Addgene plasmid # 49329), pCIG AKT3 (K177M) (Addgene plasmid # 73051), pMCL-HA-MAPKK1-8E (K97M) (Addg-ene plasmid # 40811), IRES-GFP-AXL-KD (K567R) (Addgene plasmid # 65498), and FLAG.PKCepsilon.K/W (K437W) (Addgene plasmid # 10796) were gifts from Melanie Cobb, Joseph Gleeson, Natalie Ahn, Aaron Meyer, and Alex Toker, respectively (Baek et al., 2015; Cenni et al., 2002; Meyer et al., 2015; O’Neil et al., 1990).
    HEK293T cell transfection and stimulation
    HEK293T 2-Guanidinoethylmercaptosuccinic Acid were seeded at the density of 0.7 million per well in 6-well plates. After 24 h, cells were transfected with 2 mg plasmid and 4 mL of jetPRIME (PolyPlus) per well with the standard protocol provided by the manufacturer. For kinase and phosphatase dou-ble transfection experiments, cells were transfected with plasmids encoding GFP-tagged kinases and FLAG-tagged phosphatases 16 h and 24 h after seeding, respectively. Half the amounts of plasmid and jetPRIME were used in each round for co-overexpression experiments. At 18 h after transfection, EGF (Peprotech) was added to a final concentration of 100 ng/ml. At 20 min before a given EGF stimulation time point, 5-iodo-deoxycytidine (IdU) was added to the medium at the final concentration of 10 mM. At 2 min before a given EGF stimulation time point, medium was replaced by 1X TrypLE to induce cell detachment. At the time point, paraformaldehyde (PFA, from Electron Microscopy Sciences) was added to the cell suspension to a final percentage of 1.6%, and cells were incubated at room temperature for 10 min. If EGF stimulation was not necessary in the experiment, cells were directly harvested and crosslinked with PFA.
    A375 cell transfection
    A375 cells were seeded at the density of 0.15 million per well in 6-well plates. At 24 h after seeding, transfection was performed using 2 mg plasmid and 4 mL of X-treme GENE HP reagent (06 366 236 001, Roche) per well with the standard protocol provided by the manufacturer.
    Kinase inhibition with small molecules
    BRAFV600E inhibitor vemurafenib and MEK inhibitor CI1040 (stock solutions of 10 mM/mL in DMSO, Selleckchem) were added to the cells 18 h after transfection independently or in combination at final concentrations of 1 mM and 10 mM, respectively. The same vol-ume of DMSO was added to the control samples. Cells were treated either for 3 h or for 2 days. At the end of the treatment, cells were labeled with IdU during 20-minute incubation and subsequently harvested by 5-minute TrypLE digestion and 10-minute PFA cross-linking as described above.
    Methanol permeabilization
    Crosslinked cells were washed twice with cell staining media (CSM, PBS with 0.5% BSA). After centrifugation, ice-cold methanol was used to resuspend the cells, followed by 10-minute permeabilization on ice or for long-term storage at 80 C.
    Antibody conjugation
    The MaxPAR antibody conjugation kit (Fluidigm) was used to generate isotope-labeled antibodies using the manufacturer’s standard protocol. After conjugation, the antibody yield was determined based on absorbance of 280 nm. Candor PBS Antibody Stabilization solution (Candor Bioscience GmbH) was used to dilute antibodies for long-term storage at 4 C.
    Barcoding and staining protocol
    Formalin-crosslinked and methanol-permeabilized cells were washed three times with CSM and once with PBS. Cells were incu-bated in PBS containing barcoding reagents of 89Y (100 nM), 103Rh (2 mM), 105Pd (100 nM), 106Pd (100 nM), 108Pd (100 nM), 110Pd (100 nM), 113In (200 nM), 115In (100 nM), and 209Bi (20 nM) for 30 min at room temperature and then washed three times with CSM. Barcoded cells were then pooled and stained with the metal-conjugated antibody mix (Table S2) at room temperature for 1 h. The antibody mix was removed by washing cells three times with CSM and once with PBS. For DNA staining, iridium-containing intercalator (Fluidigm) diluted in PBS with 1.6% PFA was incubated with the cells at 4 C overnight. On the day of the measurement, the intercalator solution was removed, and cells were washed with CSM, PBS, and ddH2O. After the last washing step, cells were resuspended in ddH2O and filtered through a 70-mm strainer.