Ionomycin

The Rhod-2 AM dye (4 µM, 20 min, ab142780, Abcam, UK) and Fura-2 AM dye (2 µM, 20 min, ab120873, Abcam, UK) were used to detect the mitochondrial and cytosolic Ca²⁺ level, respectively. Images were obtained every 10 s, and Ionomycin (50402ES03, Yeasen, China) was added 60 s after the beginning of the observation [39 (link)]. The relative fluorescence intensity was measured by Image J software. OCR of hepatocytes was measured by using the XF24 Extracellular Flux Analyser (Agilent SeaHorse Bioscience, USA) as described previously [40 (link)].

HK-2 cells were seeded into 384-well microplates with 8 × 10³ cells per well and grown overnight. After exposure to U at 0, 100, and 600 μM for 24 h, the cells were incubated with 25 µL of the dye loading solution containing Fluo-4 AM and probenecid at 37 °C for 30 min, then at room temperature for an additional 30 min using Fluo-4 NW Calcium Assay Kit (Thermo Fisher Scientific, #F36206) according with the manufacturer instruction. Fluorescence intensity was recorded at excitation wavelength of 470 nm and emission wavelength of 516 nm by an FLIPR Penta high-throughput real-time fluorescence imaging analysis system (FLIPR) operated by FLIPR Penta (Molecular Devices). The changes of Fluo-4 fluorescence ΔF over basal fluorescence F₀ (ΔF/F₀) were used to monitor the changes of cytosolic [Ca²⁺] upon stimulation. Ionomycin (2 μM, Yeasen Biotechnology Shanghai, China, #50401ES03) was added at the beginning of all experiments to induce Ca²⁺ release from ER Ca²⁺ stores^{77 (link),78 (link)}. ML-SA1 at 10 μM was added after Ionomycin treatment to induce the Ca²⁺ release from lysosomes.

Chemicals used in the study include afuresertib, SC-79 (Selleck, Houston, TX, USA), ionomycin (Yeasen, Shanghai, China) and Fluo-8 (Sunnyvale, CA, USA). Antibodies used in the study are NCX2 (Invitrogen, Carlsbad, CA, USA), CDH1 (Promab, Changsha, USA), Slug, Snail, AKT1, phospho-AKT1 (S473), HLA-B, ACTB, and GAPDH (ABclonal, Wuhan, China). Antagomir-193a-5p was purchased from RIBOBIO (Guangzhou, China).