Dihydroethidium dhe

DMEM, 1 × PBS, antibiotics, and FBS were obtained from GE Healthcare Life Sciences (Hyclone, Logan, UT, USA). Specific antibodies against Bcl-2 (B-cell lymphoma 2, #15071), Bax (Bcl-2-associated X protein, #2774), cytochrome c (#4272), cleaved caspase-3 (#9661), PARP (poly (ADP-ribose) polymerase, #9542), and cleaved PARP (#5625) were purchased from Cell Signaling Technology, Inc. (Beverly, MA, USA). A specific antibody against β-actin (sc-1616) was procured from Santa Cruz Biotechnology, Inc. (Dallas, TX, USA). ELISA kit for caspase-3/7 activity was obtained from Promega, Corp. (Madison, WI, USA). Ginsenoside standards were procured from ChemFaces Co., Ltd. (Wuhan, China). Dihydroethidium (DHE) was obtained from Cayman Chemical Company, Inc. (Ann Arbor, MI, USA). 2′,7′-Dichlorodihydrofluorescein diacetate (DCFDA), 3-(4,5-dimethylthiazol-2-yl)-2,5-diphenyltetrazolium bromide (MTT), tetraethylbenzimidazolylcarbocyanine iodide (JC-1), and all other chemicals were purchased from Sigma-Aldrich (St Louis, MO, USA). All other chemicals were of analytical grade.

Cytoplasmic superoxide was measured using dihydroethidium (DHE) (Cayman Chemical (Ann Arbor, USA; via Biomol GmbH, Hamburg, Germany). Cells were seeded in a 24-well plate at the density of 50,000 cells/well. At the indicated time-points, cells were washed with PBS (Gibco, Germany), and trypsinized with TriPLE Express (Gibco, Carlsbad, CA, USA, via Thermo Fischer Scientific GmbH, Dreieich, Germany). Collected cells were then incubated with 30 µM DHE dye for 10 min at room temperature, in the dark. The dye was removed by centrifugation and the cells were resuspended in colorless DMEM (Gibco, Carlsbad, CA, USA, via Thermo Fischer Scientific GmbH, Dreieich, Germany). Data acquisition was performed with Guava easyCyte HT (Luminex, MV ‘s-Hertogenbosch, The Netherlands), and analyzed with GuavaSoft 3.1.1.

Hepatocytes were pretreated with 100 ng/mL IL-11. Sixteen hr after IL-11 pretreatment, the cells were stimulated by 100 U/mL IFN-γ. Dihydroethidium (DHE) (Cayman, Ann Arbor, US) at 20 μM was added to the media 8 hr after the IFN-γ stimulation and incubated for 30 min, followed by substituting the media to PBS and microscopic observation with BZ-X710 (Keyence, Osaka, JP). Pictures were taken from the center of wells of a 96-well culture plate with a 4x objective lens. The capture area of each picture was approximately 0.13 cm². Since hepatocytes were seeded at 10000 cells/well and bottom area of each well was 0.32 cm², approximately 4000 cells were estimated to be within a picture. Fluorescence intensity of four independent pictures (approximately 16000 cells in total) was calculated for each experimental group. In the ImageJ analysis, the entire area of each picture was surrounded with a square and fluorescence within the square was quantified.

Platonin was synthesized by and obtained from Gwo Chyang Pharmaceuticals (Tainan, Taiwan; Figure 1A). Primary antibody against cleaved-caspase-3 was purchased from Abcam (Cambridge, UK). The anti-Iba1 antibody was purchased from Wako Chemicals USA (Richmond, VA, USA). The anti-NeuN antibody was from Merck Millipore (Darmstadt, Germany). For immunostaining, CF488A donkey anti-mouse IgG and CF488A donkey anti-rabbit IgG were purchased from Biotium (Fremont, CA, USA). Dihydroethidium (DHE) was purchased from Cayman Chemical (Ann Arbor, MI, USA). Platonin was dissolved in phosphate-buffered saline (PBS) and stored at 4 °C.