Fig. 3. N4 interacts with the STAT3-SH2 domain and interferes with the STAT3-EGFR and STAT3-NF-κB interactions.

A, B Molecular docking model revealed that N4 binds to the SH2 domain of STAT3. C–E The binding between N4 and STAT3 was examined in (C) SPR and (D) MST experiments. His-STAT3^127-722 protein was purified and used to examine the affinity between STAT3 and N4. (E) The binding between N4 and STAT3^SH2 was confirmed in MST assays. STAT3-SH2 protein was purified and labeled. N4 was serially diluted and mixed with equal volumes of labeled STAT3^SH2 protein. The MST signal was measured, and the data were analyzed by MO analysis software. F, G The FLAG-tag and HA-tag STAT3 expression plasmid was transfected into 293 T cells, followed by the addition of the indicated concentrations of N4. After 24 h, cells were lysed, and immunoprecipitation experiments were performed as described in Materials and Methods. H N4 represses STAT3 dimerization in pancreatic cancer cells. I After a 24 h treatment with 2 μM N4, pancreatic cancer cells were stimulated with IL-6 for 30 min. Cell lysates were immunoprecipitated with STAT3 overnight. The precipitates were washed, suspended in non-reducing sample buffer, and boiled for 10 min. The indicated antibodies were used for western blot assays. J N4 inhibits STAT3-EGFR and STAT3-NF-κB interaction in pancreatic cancer cells. PANC-1 cells were treated with 2 μM N4 for 24 h. Cell lysates were immunoprecipitated with STAT3 and blotted with the indicated antibodies.