Fig. 4. The effects of silibinin on Helicobacter pylori-induced STAT3 phosphorylation and nuclear translocation and DNA binding of STAT3.
(A) Immunocytochemistry shows the location of STAT3 protein and Hoechst staining indicates the location of the nucleus. MKN-1 cells were treated with H. pylori in the presence or absence of silibinin as described in Materials and Methods. Cells were fixed, permeabilized and stained with antibody against STAT3 followed by treatment with Alexa Fluor 488-conjugated donkey anti-rabbit IgG. Note that the STAT3 antibody used for immunocytochemustry cross-reacts with the phosphorylated form of STAT3 (P-STAT3). Nuclei were visualized by Hoechst 33258 (magnification: ×400). MKN-1 cells were treated with silibinin (100 µM) for 1 hour prior to H. pylori (500 MOI) infection for 5 hours (magnification: ×400). (B) MKN-1 cells were treated with silibinin (100 µM) for 1 hour prior to H. pylori (500 MOI) infection for 5 hours. The nuclear extracts were subjected to Western blot analysis to assess nuclear translocation of P-STAT3. (C) The infected nuclear extract cells were subjected to electrophoresis mobility shift assay to determine the nuclear translocation and DNA binging of STAT3.