Fig. 4

HMH inhibits LPS-triggered NF-κB activation. RAW 264.7 macrophages (1 × 10⁵ cells/mL) were treated with 0–10 mg/mL HMH for 2 h prior to LPS (200 ng/mL) treatment. (A) After 30 min of LPS exposure, nuclear proteins were extracted, and western blotting was performed to evaluate the expression of p50 and p65. Relative density of p50 and p65 was shown relative to nucleolin. (B) After 30 min of LPS treatment, NF-κB luciferase activity was measured using a GLOMAX luminometer and normalized with Renilla luciferase activity. (C) In a parallel experiment, cells (1 × 10⁴ cells/mL) were grown in 3 % gelatin-coated coverslips and treated with 0–10 mg/mL HMH for 2 h prior to treatment with 200 ng/mL LPS for 30 min. The cells were incubated with an anti-p65 antibody, which was captured with Alexa Fluor 488-conjugated secondary antibody. Nuclei were counterstained with 300 nM DAPI. ∗∗∗p < 0.001 vs. untreated cells and ^#p < 0.05 and ^###p < 0.001 vs. LPS-treated cells.