Figure 4.

SEA, ECM, and IPSE can activate c‐Jun and STAT3 as well as markers for DNA replication and cell cycle inhibition in vitro. (A) Concentration‐dependent expression and activation of c‐Jun by SEA was shown by western blot. (B) IF and western blot analysis of c‐Jun and p‐c‐Jun demonstrated activation of c‐Jun by SEA in Huh7 cells with maximal stimulation after 4 hours. (C) Stimulation with ECM induced a concentration‐dependent expression and phosphorylation of c‐Jun and STAT3 in Huh7 cells. (D) The SEA‐activated expression and phosphorylation of c‐Jun was inhibited by a specific inhibitor for JNK in Huh7 cells. (E) Western blot analysis for c‐Jun as well as for their phosphorylated forms showed a concentration‐dependent activation of both factors nIPSE in Huh7 cells. (F) The SEA‐induced activation of c‐Jun, MCM2, and cleavage of caspase 3 were prevented by inhibition of JNK in primary hiPS‐Hep. For control and for zero‐concentration in titration experiments, appropriate buffers of SEA, ECM, or IPSE samples were applied. The in vitro activation of c‐Jun and STAT3 fluctuated with different charges of SEA and ECM but also with different passages of Huh7 exhibiting different basal activation of the transcription factors. Nevertheless, in vitro assays were repeated at least 2‐fold, and representative blots and stainings with clear activation patterns were depicted. Abbreviations: con, control; IF, immunofluorescence.