Figure 2

Garcinol docks on to SH2 domain of STAT3 and inhibits STAT3 dimerization and acetylation, thereby preventing its nuclear localization and DNA binding. (A) Diagrammatic representation of different domains of STAT3. (B) Predicted model of garcinol binding to the STAT3 SH2 domain as shown by computational docking. Protein structure information was obtained from Protein Data Bank entry 1BG1. Garcinol shows interaction with residues Ser614, Gly617, Glu638 and Thr641 of STAT3. (C) Surface view of docked garcinol on STAT3 protein surface. (D) Garcinol inhibits STAT3 dimerization in vitro. Native protein extracts prepared from 293 T cells transfected with FLAG-STAT3 were incubated with two different concentrations of garcinol for 15 min at 30°C and were loaded in 6% SDS free native PAGE. STAT3 dimers were visualized by immune-blotting with anti-FLAG antibody. (E) Garcinol inhibits STAT3 dimerization in vivo. 293 T cells transfected with FLAG-STAT3 were treated with DMSO or garcinol (25 μM) for 3 h and whole cell lysates were prepared. Twenty micrograms of protein per sample was loaded onto a 10% native PAGE gel, and electrophoresis was performed in the absence of SDS and subjected to immune-blotting analysis. (F) Garcinol inhibits acetylation of STAT3 in cells in a dose dependent manner. HepG2 cells were incubated with 1 mM sodium butyrate (NaBu) for 6 h followed by treatment with different doses of garcinol. Cells were lysed in Laemmli buffer and immunoblotting was performed using antibodies against acetylated STAT3 and total STAT3. (G) Garcinol mediated inhibition of STAT3 acetylation reduces DNA binding ability of STAT3. FLAG-STAT3 was immuno-affinity purified from HEK293T cells (after transient transfection of FLAG-STAT3 construct for 24 h) and was incubated with garcinol in a dose dependent manner and EMSA assay was carried out using a ³²P-labelled oligo containing STAT3 binding site. For supershift validation, STAT3 antibody was added in lane 3.