Echo acoustic liquid dispenser

Proliferation assays with the 21-cell line panel (Figure 1c) and EEF1A1 mutant and parental HCT116 cells (Krastel et al., 2015 (link)) were performed using an ultra-high throughput screening system (GNF Systems, San Diego, CA). For the 21-cell line panel, cells were harvested and suspended at a concentration of 50,000 cells/mL in the appropriate medium. Cells (250 cells/well, 5 μL/well) were then dispensed into Greiner (Monroe, NC) white, solid-bottom, TC-treated, 1536-well assay plates (Griener # 789173-A) and incubated for 10 hr at 37°C (95% humidity, 5% CO₂). Compounds (15 nL/well, 16-point two-fold dilution series) and controls (15 nL/well, MG-132 and DMSO) were then added to the assay plates (n = 4) using an Echo acoustic liquid dispenser (Labcyte, Sunnyvale, CA). Assay plates were incubated 3 days at 37°C (95% humidity, 5% CO₂) before addition of CellTiter Glo (4 μL/well, Promega, Madison, WI). Assay plates were incubated for 15 min at room temperature and then luminescence was measured using a ViewLux uHTS Microplate Imager (PerkinElmer, Waltham, MA). Reported IC₅₀ values were calculated as described (Barretina et al., 2012 (link)). Experiments with EEF1A1 mutant and parental HCT116 cells were conducted in the same manner as described above, except compounds were applied by 22-point two-fold dilution series (30 μM–0.1 nM).

IRE1 endoribonuclease activity was measured using a FRET derepression assay monitoring cleavage of a 29-nucleotide stem-loop RNA containing the XBP1 cleavage site sequence and labelled with a fluorescence emitter (FAM) and a fluorescence quencher (BHQ) at the 5′ and 3′ ends, respectively [29 (link)] (Figure 7C). Upon cleavage of the stem-loop, dissociation of the product strands leads to derepression of the FRET between emitter and quencher, and signal is recovered. Briefly, varying volumes of compound in DMSO or DMSO alone were added to a low volume 384 well plate (3676, Corning, USA) to give final concentrations ranging from 100 μM to 0.313 nM using an Echo acoustic liquid dispenser (Labcyte, CA., USA). Non-phosphorylated IRE1 G547-L977 was added to a final concentration of 200 nM. After incubating for 30 minutes at 30^o C, hairpin RNA XBP-1 substrate mimic labelled with fluorescein and Black Hole quencher (5′ FAM-GAACAAGAUAUCCGCA-GCAUAUACAGUUC-3′ BHQ, Eurofins MWG Operon, Germany) was added to 100 nM final concentration. After incubating for a further 15 minutes at 30°C, the fluorescein fluorescence was measured on an EnVision plate reader (Perkin-Elmer, MA., USA). Fluorescence in the presence of compound was expressed relative to that of DMSO alone (no compound).

Compounds were diluted in 10% DMSO and dispensed at the Chemical Biology Screening Platform of the Nordic Centre for Molecular Medicine (Oslo, Norway) into individual wells of a 384-well plate using an Echo acoustic liquid dispenser (Labcyte, San Jose, CA, USA) such that, after addition of cells, the compound concentration was 5 μM. Drug libraries that were used were the Sigma LOPAC1280 library and the ChemBioNet drug library (52 (link)).
A total of 1500 cells were mixed at a BCR–Abl:WT cell ratio of 1.3:1 in 50 μl cell culture medium (containing IL3). This ratio accounted for the slower growth rate of cells transfected with BCR–Abl and ensured that the ratio after 72 h treatment with DMSO was 1:1. In addition to library compounds, imatinib (5 μM) was used as a positive control in 16 wells of each 384-well plate. After 72 h, the number of GFP-positive (BCR–Abl) and red fluorescent protein–positive (WT) cells in each well was recorded by flow cytometry and used to calculate a cell ratio. The rationale for using 72 h was that during the course of our experiments we observed that in the presence of most drugs the cells had undergone at least several doublings after this amount of time (unperturbed Ba/F3 cells have a doubling time of approximately 12 h), which is essential for identifying a potential in vitro therapeutic index.