Figure 3.

Lupeol downregulates IL-6-induced phospho-STAT3 in HCC cells. (A) The Hep3B cells (2 × 10⁶ per ml) were treated with 50 μM lupeol for the indicated times and then stimulated with IL-6 (10 ng ml⁻¹) for 15 min. One set of Hep3B cells were preincubated for 18 h in the presence of neutralising IL-6 receptor antibody (1 μg ml⁻¹) before stimulation with IL-6 to serve as a positive control. Whole-cell extracts were then prepared and analysed for p-STAT3 by western blotting. The same blots were stripped and reprobed with STAT3 antibody to verify equal protein loading. (B) The Hep3B cells (2 × 10⁶ per ml) were treated with 50 μM lupeol for the indicated times and then stimulated with IL-6 (10 ng ml⁻¹) for 15 min. One set of Hep3B cells were preincubated for 18 h in the presence of neutralising IL-6 receptor antibody (1 μg ml⁻¹) before stimulation with IL-6 to serve as a positive control. Whole-cell extracts were then prepared and analysed for p-JAK1 by western blotting. The same blots were stripped and reprobed with JAK1 antibody to verify equal protein loading. (C) The Hep3B cells (2 × 10⁶ per ml) were treated with 50 μM lupeol for the indicated times and then stimulated with IL-6 (10 ng ml⁻¹) for 15 min. One set of Hep3B cells were preincubated for 18 h in the presence of neutralising IL-6 receptor antibody (1 μg ml⁻¹) before stimulation with IL-6 to serve as a positive control. Whole-cell extracts were then prepared and analysed for p-JAK2 by western blotting. The same blots were stripped and reprobed with JAK2 antibody to verify equal protein loading. (D) The PLC/PRF5 cells (5 × 10⁵ per ml) were transfected with STAT3-luciferase (STAT3-Luc) plasmid, incubated for 48 h and treated with indicated doses of lupeol for 6 h and then stimulated with EGF (100 ng ml⁻¹) for 2 h. Whole-cell extracts were then prepared and analysed for luciferase activity. The results shown are representative of two independent experiments. *P<0.05. Abbreviation: DN=dominant negative.