Figure 4

Fluorescence-based ROS detection using the CM-H₂DCFDA probe. R. anatipestifer CH-1pLMF03, R. anatipestifer CH-1ΔfurpLMF03 and R. anatipestifer CH-1ΔfurpLMF03::fur cells were grown in GCB liquid medium with no addition (A), with 25 μM EDDHA (B) or with 50 μM EDDHA (C) at 37 °C in a shaking incubator until the exponential growth phase (OD₆₀₀ = 1.0–1.5). Then, the cells were collected and diluted in PBS to 0.5 OD/mL, and the bacterial suspensions were treated with 10 μM CM-H₂DCFDA for 30 min before treatment with 5 mM H₂O₂ or 10 mM H₂O₂ for 30 min in the dark. After exposure to H₂O₂, the cells from each culture were added to a dark 96-well plate, and the fluorescence signals were measured as described in the “Materials and methods”. The error bars represent the standard deviations of three independent experiments and three replicate samples for each experiment. Statistical significance was determined using two-way ANOVA (****P < 0.0001, *** P < 0.001).