Figure 3.

SC-43 enhances SHP-1 tyrosine phosphatase activity by targeting the autoinhibited SH2 domain of SHP-1 to suppress p-STAT3^Tyr705signaling and further induce apoptosis. (A, upper panels) SHP-1 tyrosine phosphatase activity was measured in the cells (Hct-116, DLD1, HT-29, and Hct-15) after treatment with or without 5 μM SC-43 for 24 hours. (Lower panels) SC-43 increased the SHP-1 tyrosine phosphatase activity in immunoprecipitation-SHP-1–containing cell extract. The results are shown as mean ± SD of three independent experiments, made in triplicate.*P < .05. (B) SC-43 induced a decrease in p-STAT3^Tyr705. Apoptosis occurring in Hct-15 cells was dependent on SHP-1 as assessed by using 20 nM of SHP-1 phosphatase-specific inhibitor (PTPIII) and 25 nM of siRNA specifically depleted SHP-1 as shown in the left panels and middle panels, respectively. (Right panels) Overexpression of SHP-1 in Hct-15 cells treated with 5 μM SC-43 increased the effects of SC-43 on p-STAT3^Tyr705 and apoptosis. Data from apoptosis assay are shown as mean ± SD of three independent experiments, made in triplicate.*P < .05. (C, upper panels) A schematic representation of wild-type and mutant SHP-1 (dN1 with a deletion of the N-terminal inhibitory domain, and D61A, a single point mutation). (Lower panels) Two days after Hct-116 cells were treated with 5 μM SC-43, SHP-1 activity was potently increased in wild-type SHP-1–expressing but not in dN1 or D61A mutant SHP-1–expressing cells. Data from SHP-1 activity are shown as mean ± SD of three independent experiments, made in triplicate (*P < .05; nonsignificant). (D, upper panels) Western blotting of p-STAT3^Tyr705 and SHP-1 in the wild-type and mutant-type (dN1 And D61A) SHP-1–transfected Hct-116 cells 2 days after treatment with or without SC-43 at 5 μM. (Lower panels) Apoptosis was detected in these cells as shown in upper panels of (D). Data from apoptosis assay are shown as mean ± SD of three independent experiments, made in triplicate (*P < .05; nonsignificant).