Staurosporine

To evaluate cell susceptibility to extrinsic stimuli, XFM and SCM cells (5 × 10³ cells/cm²) were seeded into 12-multiwell plates and treated with 1 μM staurosporine (Wako Pure Chemical Industries) or 100 μM hydrogen peroxide (H₂O₂; Wako Pure Chemical Industries) for 6 h at 37 °C, or were exposed to ultraviolet (UV) radiation using a UV transilluminator (UVP, Upland, CA, USA) for 30 min at room temperature. staurosporine, which is a known chemical inducer of apoptosis, was used as a positive control for cell-damage induction. Cell damage was evaluated by flow cytometry using an Annexin V/propidium iodide (PI) system described previously [28 (link)], with minor modifications. Briefly, the treated cells were stained with 100 μl of Guava Nexin® reagent (Merck KGaA) for 20 min at room temperature in the dark and subjected to flow cytometry in the Guava™ EasyCyte HT system equipped with Guava™ InCyte software (Merck KGaA). The data were analyzed with FlowJo software (TreeStar). Annexin V-positivity and PI-negativity indicated the fraction of early apoptotic cells and Annexin V/PI-double positivity indicated the fraction of late apoptotic cells. We considered that both fractions represented damaged cells [28 (link)]. The results were expressed as the ratio of the number of damaged cells to the number of nondamaged cells.

MCF-7 cells grown on coverslip were incubated with STS (24 µM) for 48 h. Cells were fixed with 4% paraformaldehyde (FUJIFILM Wako Pure Chemical Corporation) and permeabilized with 0.2% Triton X-100 (FUJIFILM Wako Pure Chemical Corporation). After blocking with 3% bovine serum albumin (BSA: Sigma-Aldrich) in PBS, cells were incubated for 1 h with primary antibodies against Proliferating Cell Nuclear Antigen (PCNA) and cleaved caspase-3 (Cell Signaling Technology, Danvers, MA, USA) and wash with 0.1% Tween 20 in PBS. Then, cells were treated with Alexa Fluor 488- and 568-conjugated goat anti-mouse and rabbit secondary antibody (Thermo Fisher Scientific). The nuclei were stained with DAPI (FUJIFILM Wako Pure Chemical Corporation). After washing, laser scanning confocal microscopy was performed using FV10i-LIV confocal microscope (Olympus Corporation, Tokyo, Japan). To quantify PCNA expression, fluorescence intensity of nuclear PCNA was quantified using ImageJ software (National Institutes of Health, Bethesda, MD, USA). Staurosporine (FUJIFILM Wako Pure Chemical Corporation) was used as the positive control for apoptosis.

To generate iNTCP cells, a HepG2 Tet-On Advanced Cell (Clontech) parental cell line was transduced with a retroviral vector encoding the NTCP gene fused to a tetracycline-responsive element, then was selected with puromycin (1 μg/ml), and cultured with DMEM (Wako) supplemented with 10% FBS. Unless otherwise indicated, iNTCP cells were treated with doxycycline (Sigma-Aldrich) for 24 hours before experiments. The iNTCP spheroid was made using the 3-D Life Dextran-CD Hydrogel SG kit (Cellendes) and cultured for seven days on a chamber slide (Thermo Fisher Scientific). Primary human hepatocytes (PXB-cells) were purchased from PhoenixBio. HepaRG cells were purchased from Biopredic International and differentiated according to the manufacturer's instructions. The chemical compound library from natural plant extracts was obtained from Tokiwa Phytochemical. Nocodazole, cyclosporin A, genistein, and Pitstop2 were purchased from Sigma-Aldrich. Glabridin, staurosporine, and IFN-β were obtained from Wako.