Fig. 2
Genipin directly binds with STAT-3-SH2 domain. (a) 3D docking results indicated that genipin could bind to SH2 domain in STAT-3. (b) Purified GST-tagged STAT-3-SH2 protein was analyzed by SDS-PAGE. Coomassie blue was used to stain the target protein. (c) Fitted curve for different concentrations of genipin binding to immobilized STAT-3-SH2 by “Affinity” model in the Biocore T300 evaluation software. Differently colored dots represent different concentrations of genipin. (d) Genipin-suppressed SH2-SH2 interactions. Purified STAT-3-SH2 domain was incubated with MHCC97L cell lysates after mixing with different dosages of genipin for 1.5 h and analyzed by GST pull-down assay. (e) Genipin suppressed the dimerization of STAT-3. MHCC97L cells transfected with HA and FLAG-tagged STAT-3 vectors were pre-treated with genipin, the interaction of FLAG-STAT-3 and HA-STAT-3 were validated by immunoprecipitation assay. (f) Purified STAT-3-SH2 domain was incubated with MHCC97L cell lysates after mixing with different dosages of genipin for 1.5 h; the interactions of STAT-3-SH2 with EGFR were validated by GST pull-down. (g) HCC cells (MHCC97L and HepG2) were pre-treated with different dosages of genipin and incubated with EGF; the interactions of STAT-3 with EGFR were validated by co-immunoprecipitation