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. 2018 Dec 12;9:1441. doi: 10.3389/fphar.2018.01441

FIGURE 6.

FIGURE 6

Effect of ZCO on NF-κB transcription and translocation. (A) Effect of ZCO on PMA-induced NF-κB activation in 293T cells. 293T cells seeded into poly-D-lysine hydrobromide coated 96-well plate were transfected with a reporter plasmid, pNF-κB-SEAP for 4 h. Cells were treated with ZCO (0.0025, 0.005, and 0.01%) or Bay11-7082 (20 μM) for 2 h prior to stimulation with 3 ng/mL of PMA for 24 h. The supernatants were collected for NF-κB activity assay. Experiments were conducted in quadruplicate and expressed as mean ± SEM (∗∗∗P < 0.001 versus with PMA group). (B) Effect of ZCO on NF-κB translocation into nucleus. RAW264.7 cells in 8-well glass chamber plate were pre-incubated with ZCO (0.01%) or Bay11-7082 (20 μM) for 2 h prior to addition of LPS (1 μg/mL). The cells were immunostained with a polyclonal anti- NF-κB p65 antibody and Alexa Fluor 488-conjugated 2nd antibody. Bay 11-7082 was used as a control for the NF-κB inhibitor. (C) Cytosolic and nuclear fractions were isolated from RAW264.7 cells. The protein level of NF-κB p65 in the nuclear fractions and IkB-α in the cytosolic fractions were detected by Western blot analysis. +++means the significance compared with the mock cells (deal with nothing).