Jc1 staining solution

The cells were seeded into cell culture plates and stained with JC1 staining solution (40705ES03, Yeasen Biotechnology, Shanghai, China) following the instruction manual. The fluorescence was observed with Leica DMi8 fluorescence microscope. Reactive Oxygen Species were detected using the Reactive Oxygen Species Assay Kit (50101ES01, Yeasen Biotechnology, Shanghai, China), and visualized with Leica DMi8 fluorescence microscope. The quantification of fluorescence results was done using the Image J software. Additionally, ER-Tracker Green (BODIPY FL Glibenclamide) (40763ES20, Yeasen Biotechnology, Shanghai, China), ER-Tracker Blue-White DPX (40761ES50, Yeasen Biotechnology, Shanghai, China) and MitoTracker Red CMXRos (40741ES50, Yeasen Biotechnology, Shanghai, China) were used to stain the endoplasmic reticulum or mitochondria of live cells. The staining was visualized using laser scanning confocal microscopy (Leica, TCS SP8 DIVE, Germany), and the co-localization was quantified using the co-localization Finder plugin of ImageJ software. The experimental protocols were conducted as per the standards.

Cells were trypsinized and resuspended in 1X binding buffer, and stained using the Annexin-V-FITC/PI Apoptosis Detection kit (40305ES50, Yeasen Biotechnology, Shanghai, China). The cells were then washed with PBS, and cell apoptosis was analyzed using flow cytometry (FACSCalibur, BD, Biosciences, USA). Mitochondrial membrane potential detection: Cells were tripsinized and resuspended in 1X binding buffer, and stained using the JC1 staining solution (40705ES03, Yeasen Biotechnology, Shanghai, China) following the instruction manual. The cells were re-suspended with fresh culture solution for subsequent flow cytometry analysis. Reactive oxygen species (ROS) detection: DCFH-DA (50101ES01,Yeasen Biotechnology, Shanghai, China)was diluted in serum-free medium at 1:1000 to a final concentration of 10 μM; Cells were tripsinized and resuspended in appropriate volume of diluted DCFH-DA working solution. After staining, the cells were re-suspended with fresh serum-free medium and immediately detected by flow cytometry.

The apoptosis analysis was performed using the Annexin V-FITC/PI Apoptosis Detection Kit (KeyGEN BioTech, Nanjing, China) following the aforementioned method [28 (link)]. The excitation (Ex) and emission (Em) wavelengths are 488 nm and 530 nm, separately. The FITC positive/PI positive and FITC positive/PI negative cells were considered as apoptotic cells. The negative control was shown in Additional file 2: Fig. S1. The mitochondrial membrane potential (ΔΨm) change was also determined by flow cytometry. Briefly, cells were collected and incubated with 10 µg/mL JC-1 staining solution (Yeasen, Shanghai, China) for 15 min at a cell culture incubator. Then, cells were analyzed by flow cytometry immediately (Ex = 488 nm, Em = 530 nm). The rate of Q2 quadrant represented no loss of ΔΨm.