Figure 5.

EspC-induced ER stress response is involved in mitochondrial apoptosis. (A-C) RAW264.7 cells were treated with PBS, EspC, or Ag85A for varying durations or 36 h. Cells were collected and stained with DiOC6 (10 nM), and ΔΨm was evaluated using flow cytometry (D) RAW264.7 cells were stimulated with EspC (5 µg/mL) or Ag85A for 24 h, and the expression levels of Bax and cytochrome c were analyzed using qPCR. (E) RAW264.7 cells were stimulated with EspC, Ag85A (5 µg/mL), or PBS for 36 h. Subcellular fractions of the mitochondria and cytoplasm were extracted and analyzed using western blot analysis with antibodies against Bax, cytochrome c (Cyt C), COX IV, and α-tub1. COX IV and α-tub1 were used as markers of mitochondrial and cytosolic fractions, respectively. (F) Representative confocal laser scanning microscopy images showing the Bax translocation from the cytosol into the mitochondria and the Cyt C release from the mitochondria to the cytosol in EspC-stimulated RAW264.7 cells. Scale bar = 5 µm or 2.5 µm. (G) RAW264.7 cells were pretreated with 4-PBA (2 mM) for 1 h and incubated with EspC for 36 h. Subcellular fractions of the mitochondria and cytoplasm were extracted and analyzed using western blot analysis with antibodies against Bax, cytochrome c (Cyt C), COX IV, and α-tub1. Data are shown as the mean ± SEM (n = 3); * p < 0.05, *** p < 0.001.