Fig 1: Effect of 14 on SHP2 activation. (A) UMR106 cells treated with compounds for 30 min were lysed and immunoprecipitated with the anti-SHP2 antibody. Thereafter, SHP2 activity was determined. GAPDH was used as the input. (*) p < 0.05 and (****) p < 0.0001 compared to the DMSO control. (B) Time-dependent effect of 14 on SHP2 activity. UMR106 cells treated with 10 µM NSC-87877 (30 min) and 10 µM 14 at the indicated time were lysed, immunoprecipitated with anti-SHP2 antibody, and SHP2 activity was determined. (*) p < 0.05, (**) p < 0.01 and (***) p < 0.001 compared to the DMSO control. (C) UMR106 and MC3T3-E1 cells were treated with compounds for 1 h. The cell lysates were immunoblotted with antibodies against phospho-ERK, ERK, phospho-Src-pTyr418, and Src. (*) p <0.05, (***) p < 0.001 and (****) p < 0.0001 compared to the DMSO control; (##) p < 0.01, (###) p < 0.001, (####) p < 0.0001 compared to compared to 14 alone treatment. (D) UMR106 and MC3T3-E1 cells were treated with 14 (10 µM) or sildenafil (10 µM) alone for 24 h, or pretreated with NSC-87877 for 1 h, and then 14 (10 µM) was added for additional 24 h. Cell lysates were immunoblotted with antibodies against aromatase. GAPDH was used as the internal control. (*) p < 0.05, (**) p < 0.01, (***) p < 0.001 compared to the DMSO control. (###) p < 0.001 compared to 14 alone treatment. Cont., DMSO-treated control; NSC, 10 µM NSC-87877; Sil, 10 µM sildenafil. Error bars represent the standard deviation of the measurement.