Fig 1: MIC-1 selectively inhibits PTP1B activity. (A) The inhibitory activities of MIC-1 and suramin against PTP1B. (B) The inhibitory activity of MIC-1 against TC-PTP. (C) MIC-1 directly binds to PTP1B with high affinity (KD = 2.0 × 10−5 M). Biolayer interferometry (BLI) sensorgrams indicating the interactions between gradient concentrations of MIC-1 and PTP1B were measured on an Octet Red96 system, with association and dissociation for 90 s each. (D) Three-dimensional simulation of the interaction between MIC-1 and the PTP1B protein. (E) Two-dimensional simulation of the interaction between MIC-1 and the PTP1B protein. Values represent the means ± SEM from three independent experiments and statistical analysis was performed by one-way ANOVA; **p < 0.01, ***p < 0.001: MIC-1 compared with the control (0 μM).
Fig 2: Effect of MIC-1 on PTP1B-related Src/Ras/Raf/ERK signaling pathway in non-renal cancer cells. (A) HCT-116 cells, Hep-G2 cells, and A431 cells were treated with MIC-1 (0 or 4 μM) for 48 h, and the expression of Src, p-Src (Tyr416), K-Ras, ERK1/2, and p-ERK1/2 was detected by western blotting. β-actin served as a loading control. Quantification of the relative levels of p-Src (Tyr416) (B), K-Ras (C), and p-ERK1/2 (D); each value was normalized to that of Src, β-actin, and ERK1/2, respectively. Values represent the means ± SEM from three independent experiments and statistical analysis was performed by unpaired two-tailed Student’s t tests.
Fig 3: MIC-1 inhibits the growth and migration of 786-O cells by inhibiting the PTP1B-mediated activation of the Src/Ras/Raf/ERK signaling pathway. (A) 786-O cells were treated with MIC-1 (0, 2, or 4 μM) for 48 h, and the expression of Src, p-Src (Tyr416), p-Src (Tyr529), K-Ras, Raf, p-Raf, ERK1/2, and p-ERK1/2 was detected by western blotting. β-actin served as a loading control. (B) Detection of PTP1B mRNA and protein expression in 786-O cells transfected with a PTP1B cDNA expression vector by RT-PCR and western blotting, respectively. (C) Untransfected and PTP1B-overexpressing 786-O cells were treated with different concentrations of MIC-1 (0, 2, or 4 μM) for 48 h after which cell viability was analyzed by the MTT assay. (D) Clonogenic ability was analyzed by a colony formation assay. (E) Untransfected and PTP1B-overexpressing 786-O cells were treated with different concentrations of MIC-1 (0, 1, or 2 μM) for 24 h after which migratory ability was analyzed using a wound healing assay. (F) Untransfected and PTP1B-overexpressing 786-O cells were treated with MIC-1 (0, 2, or 4 μM) for 48 h, and the expression of Src, p-Src (Tyr416), p-Src (Tyr529), ERK1/2, and p-ERK1/2 was detected by western blotting. β-actin served as a loading control. Values represent the means ± SEM from three independent experiments and statistical analysis was performed by one-way ANOVA; *p < 0.05, **p < 0.01, ***p < 0.001: MIC-1-treated cells compared with control 786-O cells (0 μM) or PTP1B-overexpressing 786-O cells (0 μM); PTP1B-overexpressing 786-O cells compared with control 786-O cells.
Fig 4: Inhibition of PTP1B enzymatic activity by suramin and ondansetron (gray bars) at different concentrations. The X-axis represents the increasing concentrations of each compound, while the y-axis shows the % activity of the PTP1B enzyme.
Supplier Page from Abcam for PTP1B Inhibitor Screening Assay Kit