Figure 6. Fucoidan induces intracellular ROS generation in A549 cells.

(A) A549 cells (5 × 10⁴ cells per well in 96-well plates) were treated with fucoidan (400 μg/ml) or NAC (5 mM) for 0–60 min and then stained with fluorescent dye (H₂DCFHDA, DCF) for 30 min. The fluorescence intensity of DCF was detected using Infinite 200 PRO multimode microplate readers (TECAN, Switzerland) at excitation and emission wavelengths of 485 and 535 nm, respectively. (B,C) A549 cells were pre-treated with NAC (20 mM) for 30 min, followed by additional incubation with fucoidan (400 μg/ml) for 30 min (B) and 24 h (C). The fluorescence intensity of DCF was detected by flow cytometry. (D) A549 cells were pre-treated with 5 mM of NAC for 1 h and were then treated with fucoidan (400 μg/ml) for 24 h, and Western blotting analyses of whole-cell lysates were subsequently performed to measure ATF4 and CHOP expressions. (E) A549 cells (scramble and sh-TLR4) were treated with fucoidan (400 to 1600 μg/ml) or NAC (20 mM) for 30 min. The fluorescence intensity of DCF was detected by flow cytometry. (F) Quantification of MFI in an experiment (E) is representative of three separate determinations by FlowJo. (G) A549 cells (scramble and TLR4-knockdown cells) were treated with fucoidan (0 to 400 μg/ml) for 24 h. Cell viabilities were determined with crystal violet staining assays. Western blot analysis was performed to determine the TLR4 and CHOP protein levels.