Mitospy red cmxros

RAW264.7 cells were plated overnight on 20-mm glass-bottomed cell culture dishes. After treatment with EspC, the cells were incubated with the pre-warmed medium containing 250 nM of MitoSpy Red CMXRos (Biolegend) for 20 min. After the cells were washed with PBS, they were fixed and permeabilized in Immunol Staining Fix solution (Biyuntian) for 20 min and blocked with 3% bovine serum albumin (BSA) in PBS. The cells were washed with PBS and incubated with anti-cytochrome c and anti-Bax for 2 h at 37 °C in the Immunol staining primary antibody dilution buffer (Biyuntian). After the cells were washed with PBS, they were re-stained with the appropriate Alexa Fluor 488-conjugated secondary antibodies for 1 h at 37 °C and stained with 0.1 µg/mL DAPI for 10 min at 25 °C. The cells were analyzed using a Leica TCS SP8 laser scanning confocal microscope (Leica Microsystems, Wetzlar, Germany) (63X; NA, 1.4) oil immersion lens (HC PL APO CS2; zoom, 2.5X; speed, 400 Hz; line average and resolution, 4). Images were acquired and processed using the LAS AF Lite software.

Cells were stained with 20 nM MitoSpy Red CMXRos (Biolegend #424801) in media for mitochondrial membrane potential in live cells and 200 nM in fixed cells at 37 °C. Cells were stained with 50 nM MitoSpy Green FM (Biolegend #424806) for mitochondrial mass in live cells at 37 °C.

The IPEC-J2 cell was a courtesy from Prof. Yin Yulong, Institute of Subtropical Agriculture, Chinese Academy of Sciences. H₂O₂ (Sinopharm Group, China), DMEM-F12 medium (Shanghai Yuanpei Biotechnology, China), and fluorescein isothiocyanate dextran 4 kDa (FD4) were obtained from Sigma-Aldrich (St. Louis, Missouri, USA). Trypsin (Beyotime Biotechnology, China), phosphate-buffered saline (PBS) (Bozan Biotechnology, China), penicillin-streptomycin (Solabao Biotechnology, China), fetal bovine serum (Gemini, Australia), CCK-8 kit (Beyotime Biotechnology, China), MitoSpy™ Red CMXRos (Biolegend, USA), immunofluorescence fixative (Sevier Biotechnology, China), Triton-X 100 (Sigma-Aldrich, St. Louis, MO, USA), Glycine (Sinopharm Group, China), DAPI (Beyotime Biotechnology, China), Goat Anti-Mouse IgG Dylight 594 (Earthox, USA), Goat Anti-Mouse IgG Dylight 488 (Earthox, USA), mitochondrial division inhibitor (Mdivi-1) (Selleck, USA), AMPK inhibitor (Compound C, CC) (Selleck, USA), Lipofectamine RNAiMAX, and Lipofectamine 2000 were obtained from Invitrogen (Invitrogen, USA).