Mitosox

Without specific indication, the reagents purchased from Sigma‐Aldrich Inc. were used in the present study, and cell culture supplements were from GIBCO/Life Technologies Inc. Vincristine (VCR), SZLP1‐41 (Skp2 inhibitor), E64D (lysosomal protease inhibitor) and epoxomicin (proteasome inhibitor) were the products of Apexbio Technology LLC. GKT137831 and GLX351322 were purchased from MedChem Express; MitoSOX, annexin V‐FITC/propidium iodide (PI) apoptosis detection kit, tetramethylrhodamine (TMRM) and H₂DCFDA were from Molecular Probes (Eugene, OR). Antibodies against caspase‐3 and caspase‐8, okadaic acid, Z‐IETD‐FMK and Z‐DEVD‐FMK were purchased from Calbiochem. Antibodies separately against PP2Ac, Mcl‐1, tristetraprolin (TTP), Fas, FasL, TNF‐α and FADD were purchased from Santa Cruz Biotechnology Inc.; and antibody against TNFR1 was from R&D Systems. Antibodies separately against ERK, p‐ERK, p38 MAPK, p‐p38 MAPK, JNK, p‐JNK, SIRT3, TNFR2, α‐tubulin, MID1, α4, caspase‐9, Bax, Bak and Bcl‐2 were obtained from Cell Signaling Technology. Antibodies separately against cytochrome c, Bcl‐xL and Bid were the products of BD Pharmingen Technical (San Jose, CA), and anti‐NOX4 antibody was from Novus Biologicals. Secondary antibodies conjugated with horseradish peroxidase (HRP) were the products of Pierce (Rockford, IL).

Changes in intracellular ROS, MMP, mtROS, and mitochondrial morphology were assessed according to the manufacturer’s instructions. At the end of the treatment, the original culture medium was aspirated and treated with 2′,7′-dichlorofluorescein diacetate (DCFH-DA) (Beyotime Biotechnology, Shanghai, China), JC-1 working solution (Beyotime Biotechnology, Shanghai, China), MitoSOX (MedChemExpress, Shanghai, China) and Mito-Tracker Red CMXRos (Beyotime Biotechnology, Shanghai, China) were stained at 37 °C for 20 min. Then, cells were washed three times with DMEM/F12, and the ROS levels, MMP, mtROS, and mitochondrial morphology were observed by fluorescence microscopy (Olympus, Tokyo, Japan).

MMP values of macrophages were determined using the JC‐1 MMP assay kit (MedChemExpress, USA). Cells were incubated using jc‐1 staining working solution for 20 min at 37 °C and subsequently washed twice with PBS. Images were captured with a fluorescence microscope (Carl Zeiss, USA). MMP (ratio of red/green fluorescence intensity) was calculated using ImageJ software.
The intracellular and mitochondrial ROS were determined using DCFH‐DA (MedChemExpress, USA) and MitoSox (MedChemExpress, USA) probe. Cells were incubated with working solution with 5 × 10⁻⁶m DCFH‐DA or 5 × 10⁻³m MitoSox for 30 min at 37 °C. They were subsequently examined by fluorescence microscopy and flow cytometry respectively as mentioned above.