Fig 2: PTP1B knockdown inhibits ox-LDL-induced inflammatory injury of HUVECs. (A) The levels of IL-6, IL-1ß and TNF-a were detected using ELISA. (B) Cell apoptosis was measured by TUNEL assay. Scale bar, 50 µm. (C) Histogram of the quantified apoptosis rate of HUVECs. (D) Protein expression levels of Bcl-2, Bax, cleaved caspase-3 and caspase-3 were measured and semi-quantified via western blotting. Results represent the mean ± SD. **P<0.01 and ***P<0.001. PTP1B, protein tyrosine phosphatase 1B; ox-LDL, oxidized low-density lipoprotein; sh, short-hairpin; NC, negative control.

Fig 3: Knockdown of PTP1B expression restores the viability of ox-LDL-induced HUVECs. (A) mRNA and (B) protein expression level of PTP1B in HUVECs with or without ox-LDL treatment were detected by RT-qPCR and western blotting assays. (C) mRNA and (D) protein expression levels of PTP1B in HUVECs transfected with or without sh-PTP1B were detected by RT-qPCR and western blotting assays. (E) Cell viability was measured using Cell Counting Kit-8 assay. (F) LDH level was analyzed using a corresponding kit. Results represent the mean ± SD. **P<0.01 and ***P<0.001. PTP1B, protein tyrosine phosphatase 1B; ox-LDL, oxidized low-density lipoprotein; RT-qPCR, reverse transcription-quantitative PCR; sh, short-hairpin; LDH, lactate dehydrogenase; NC, negative control.

Fig 4: 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. ß-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, ß-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 5: 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).