EF24 induced cell cycle arrest and inhibited the NF-κB pathway.

(A). DNA content (propidium iodide) and cell cycle analysis of EF24-treated cells. The five HCC cells were incubated with 0, 2 µmol/L EF24 for 48 h. The numbers of cells in G0/G1 phase, S phase and G2/M phase was determined via flow cytometry. (B). Representative histograms from cytometrically analyzed the five cell lines treated with control and EF24. Assay was done in triplicate and p<0.05 is denoted by “*”. (C). Expression of G₂/M cell cycle relative proteins Tyr15 and Thr161 phosphorylation of cdc2 and Cyclin B1 were determined via western blot after treatment with 2 and 4 µmol/L EF24 for 24 h. β-actin was used as the internal control. (D). Nuclear extracts were prepared and subjected to EMSA to measure NF-κB DNA-binding activity. (E). Expression of p65, Bcl-2, Bax and COX-2 via western blot.