Figure 4

SHP2 acts on Snail/Twist1 through negatively regulating ERK1/2 activity. (A) SHP2 forms a complex with ERK1/2. Total cell lysates were prepared, and SHP2 was immunoprecipitated from HSC3 cells expressing EGFP-tagged SHP2 wild type or catalytic-defective SHP2 (SHP2C/S). SHP2 in association with active ERK1/2 in these cells was detected by SDS-PAGE and immunoblotting with anti-phospho-ERK1/2, ERK1/2, SHP2 and GFP. (B) Nuclear localization of phospho-ERK1/2 is enriched in HSC3-Inv4 and HSC3-Inv 8 compared to HSC3 parental cells. (C) Treatment of ERK inhibitor with indicated concentration for 6 hours significantly reduced Snail or Twist1 mRNA expression in HSC3 parental and HSC3-Inv8 cells. (D) SHP2 depletion significantly increased Snail orTwist1 mRNA expression in HSC3 parental and HSC3-Inv8 cells (Upper panel and lower panel, respectively.). Experiments were done in triplicate and values indicated as mean ± SD. *, P < 0.05 compared with adjacent normal in each case. (E) Knockdown of SHP2 increases both cytosol and nuclear localization of phospho-ERK1/2 in oral cancer cells. Poly ADP-ribose polymerase (PARP) was used as a nuclear marker.