Jc 1 fluorescent dye

Mitochondrial membrane potential (ΔΨm) was detected by JC-1 staining. Vero E6 and Huh-7 cells were seeded on coverslips at a density of 1 × 10⁵ cells/well in 12-well plates. Mock- or SARS-CoV-2-infected Vero E6 and Huh-7 cells were cultured for an indicated time. At 3, 6, and 12 h post infection, cells were washed with PBS for three times. 10 μg/ml JC-1 fluorescent dye (Sigma Aldrich, T4069) was added and incubated for 15 min. Images were captured by fluorescence microscopy.
Mitochondria permeability transition pore (MPTP) opening was assessed by the tetramethylrhodamine methyl ester (TMRM) probe (Sigma Aldrich; T5428). At 3, 6, and 12 h post infection, Vero E6 and Huh-7 cells were washed with PBS for three times. 300 nM TMRM was added and incubated for 15 min. Images were captured by fluorescence microscopy.
Reactive Oxygen Species (ROS) release was detected by the carboxy-H2DCFDA probe (Invitrogen, I36007). At 3, 6, and 12 h post infection, Vero E6 and Huh-7 cells were washed with PBS for three times. 25 μM carboxy-H2DCFDA working solution were added and incubated for 30 min. Cells were then returned to pre-warmed growth medium and incubate at 37°C for another 1 h. Images were captured by fluorescence microscopy. After the above steps, flow cytometry can also be performed to observe the changes in ROS levels.

LCT-3d was synthesized in our group and was dissolved in DMSO. RPMI-1640, Fetal bovine serum (FBS), and penicillin-streptomycin were purchased from HyClone (Victoria, Australia). DCFH-DA (2, 7-dichlorodihydrofuorescein diacetate), the Nuclear and Cytoplasmic Protein Extraction Kit, and Annexin V- FITC Apoptosis Detection Kit were purchased from Beyotime Biotechnology (Shanghai, China). The primary antibodies for Caspase 3 (sc-7272), PARP (sc-7150), DR5 (sc-65314), Caspase 9 (sc-7885), Bim (sc-11425), Bad (sc-8044), Bax (sc-493), DR4 (sc-7863), and β-actin (sc-1615) were purchased from Santa Cruz Biotechnology (Santa Cruz, CA, USA). Bid (#2002), Bcl-xL (#2764), and XIAP (#14334) were purchased from Cell Signaling Technology (Danvers, MA, USA). The secondary antibodies anti-goat, anti-rabbit, and anti-mouse were purchased from Bioss (Shanghai, China). The pan-Caspase inhibitor Z-VAD-FMK was purchased from Selleck. The Nrf2 inhibitor ML385 was purchased from MCE (NJ, USA). The ECL (enhanced chemiluminescence) kit was purchased from Thermo Fisher (Waltham, MA, USA). Anti-DR5 (ab1675) antibody for flow cytometry was purchased from Abcam (Cambridge, MA, USA). NAC (N-acetyl-L-cysteine), MTT [3-(4, 5-dimethylthiazol-2-yl) - 2, 5-diphenyltetra- zolium bromide], and JC-1 fluorescent dye were purchased from Sigma-Aldrich (St. Louis, MO, USA).