*4.8. Confocal Microscopy Analysis*

RAW264.7 cells were grown on glass coverslips. After the treatment with LPS (200 ng/mL) with or without pre-treatment with coelonin or NF-κB-inhibitor APDC, cells were fixed with 4% formalin in PBS for 20 min at room temperature, permeabilized with 0.1% Triton X-100 for 15 min, and nonspecific protein binding sites were blocked with 10% FBS at room temperature for 1 h. Then samples were incubated overnight at 4 ◦C with a p65 (ab32536, Abcam, Cambridge, MA, USA) specific Antibody (1:100 dilution in PBS), washed three times with PBS, and then incubated with a Alexa Fluor® 488-labeled secondary antibody (ab60314, Abcam; 1:600 dilution in PBS) for 2 h at room temperature. The coverslips were rinsed three times with PBS, then stained with 4 ,6-diamidino-2-phenylindole (DAPI) for 10 min. Again, the coverslips were rinsed with PBS three times and then mounted on glass slides using Antifluorescence Quenching Sealing Solution. The coverslips were analyzed by confocal microscopy (LSM880; ZEISS, Upper Cohen, Germany).

#### *4.9. Cell Cycle Analysis*

After treatment, the RAW264.7 cells were washed three times with chilled 1 × PBS (pH 7.4) and fixed overnight with 70% ethanol at 4 ◦C followed by centrifugation at 2,000 rpm for 8 min. The cells were re-suspended in 1 × PBS (pH 7.4) with PI/RNase Staining Buffer (BD, 550825, San Diego, CA, USA) for 30 min. The cell cycle distributions were analyzed on a BD Accuri™ C6 flow cytometer (BD, Ann Arbor, MI, USA).
