Synergy neo

hERG binding assay was performed using hERG Fluorescence Polarization Assay (Invitrogen: PV5365) assay kit and Synergy Neo (Biotek) and standard procedures were applied.

The in vitro kinase assay (HTRF KinEASE-STK S1, CisBio 62ST1PEB) was optimized to the linear reaction range of the enzymes Lats1 (Carna 01-123) and Lats2 (Carna 01-124). Reactions were conducted with 10 μM STK1 substrate and 10 μM ATP, unless otherwise indicated. For the ATP-shift assay, ATP was also used at concentrations of 50 and 250 μM. Lats1 was employed at a concentration of 200 pg/μL and Lats2 at 50 pg/μL unless otherwise indicated. For the assay in which Michaelis–Menten constants for ATP were determined, each enzyme was at a concentration of 62.5 pg/μL; Lats1 ran for 30 min, and Lats2 ran for 20 min, all at room temperature. The DMSO concentration was maintained at 0.5% (vol/vol) throughout all experiments, and a Janus 384 MDT (PerkinElmer) equipped with a 50 nL Pintool (V&P Scientific, Inc.) was used to add the compounds dissolved in DMSO to the reaction. The enzyme, substrate, ATP, and TRULI were combined in a low-volume 384-well plate and shaken for 50 min at room temperature unless otherwise indicated. The reaction was stopped by adding the detection reagents, which were prepared at an 8:1 biotin:streptavidin ratio, and shaken for 60 min at room temperature. All reactions were conducted in triplicate and a Synergy NEO (Biotek) was used to detect the signal.

Necrosis in vitro was induced by stimulation of oxidative stress with H₂O₂ or calcium overload with ionomycin. Plasma membrane permeabilization was determined by PI exclusion and LDH release assays. In brief, cardiomyocytes were incubated with PI (P21493, Thermo Fisher, 2 μg/ml) for 30 min at 37 °C. Necrotic cells (PI⁺) were examined under a fluorescent microscope. LDH assay was performed using the Cytotoxicity Detection Kit (LDH) (11644793001, Roche) according to the manufacturer's instructions. In addition, cell viability was also assessed using Cell Proliferation Kit I (MTT, Roche). Both LDH and MTT assays were measured with Synergy NEO microplate reader (Biotek).

Cell migration assays were performed using Transwell Permeable Supports (Costar Corning, NY, USA) with a pore size of 8.0 um, according to the Boyden chamber method. AGS-1 and HGC-27 cells seeded on the upper chamber supplemented with RPMI-1640 (serum-free) were pre-treated with 20 μM ISO. The lower chamber was supplemented with normal RPMI-1640 medium. After 24 h of incubation, the cells that migrated into the lower chamber were stained with 0.5% crystal violet (30 min) and observed using an Olympus IX73. The cells were further eluted by 50% ethanol and 0.1% acetic acid and detected by BioTek Synergy NEO at 550 nm.