FIGURE 3.

ROS generation and apoptosis of Caco-2 cells cultured in conditioned medium with and without pre-treatment of ROS inhibitor NAC. (A) Fluorescence microscopy of live Caco-2 cells untreated (control) or treated with conditioned medium with or without NAC (100 μM) pre-treatment. Carboxy-H₂DCFDA was loaded to detect H₂O₂ in live cells (green color), and Hoechst 33342 was used for nucleic acid stain (blue color). (B) Cell viability of Caco-2 cells cultured in conditioned medium ACCM (a) and TACCM (b) for 24 h with or without NAC (100 μM) pre-treatment. (C) Relative H₂O₂ generation levels of Caco-2 cells untreated (control) or treated with conditioned media with or without NAC (100 μM) pre-treatment. (D) Western blots show the effects of NAC (100 μM) pre-treatment on the expression of proteins in conditioned media-treated Caco-2 cells. Red color specifies the statistical significance as compared to the control. ROS, Reactive Oxygen Species; NAC, ROS inhibitor N-acetyl-l-cysteine. CM, fresh bacterial culture medium; TCM, Turicibacter culture medium; ACCM, fresh medium with AC; TACCM, Turicibacter culture medium with AC. Data was presented as mean ± SEM, n = 3. *p < 0.05, **p < 0.01, ***p < 0.001, as compared to the control; ^# p < 0.05 indicates the impact of NAC. Abbreviation: w/, with; w/o, without.