Figure 4.

EspC-mediated ER stress is associated with ROS production. (A, B) Intracellular hydrogen peroxide (A) and superoxide (B) levels were evaluated using flow cytometry with DCFH-DA (10 µM) for hydrogen peroxide and dihydroethidium (DHE; 20 µM) for superoxide, after treatment with EspC or Ag85A for 24 h. LPS treatment was used as the positive control. (C) RAW264.7 cells were pretreated with 4-PBA (2 mM) for 1 h and then incubated with EspC for 30 h. Cells were collected and stained with DHE, following which superoxide production was detected using flow cytometry. (D–G) RAW264.7 cells were pretreated with NAC (5, 10 µM) for 1 h and then incubated with EspC for 30 h. (D) Cells were collected and stained with DHE, following which superoxide production was detected using flow cytometry. (E) Total cell lysates were subjected to western blot analysis with specific antibodies against each target protein. (F) Apoptotic cells were quantified using flow cytometry. (G) Pro-inflammatory cytokine (TNFα, IL-6, MCP-1) levels in the supernatant were measured using ELISA. (H) RAW264.7 cells were pretreated with NAC (10 µM) for 1 h and then incubated with EspC for 24 h. Total cell lysates were subjected to western blot analysis with specific antibodies against each target protein. (I) RAW264.7 cells were pretreated with BAPTA-AM (5 µM) for 1 h and then incubated with EspC (5 µg/mL) for 24 h. Cells were collected and stained with DHE, and superoxide production was detected using flow cytometry. (J) RAW264.7 cells were preincubated with or without NAC (10 µM) for 1 h, followed by EspC treatment for 30 h. The percentage of intracellular Ca²⁺ was measured with fluo-3/AM using flow cytometry. LPS treatment was used as the positive control to induce ER stress. Data are shown as the mean ± SEM (n = 3); * p < 0.05, ** p < 0.01, *** p < 0.001.