Fig 1: The PPS effect is electrostatic(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) in the absence (○) or presence (●) of 10 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (B) TIMP-3 (0.5 nM) was incubated with ADAMTS5-5 (0.5 nM) in the absence (○) or presence (●) of 100 nM PPS and 50–500 mM NaCl (1 h, 37°C), and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C). (C) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and PPS (10 nM) in TNC buffer (containing 250 mM NaCl) at 37°C for times ranging between 1 and 72 h. The concentration of NaCl was then increased to 400 mM and residual activity against Abz-TESE~SRGAIY-Dpa-KK was determined (18 h, 37°C).
Fig 2: Longer PPS molecules are more effective than short PPS molecules at improving affinity(A) TIMP-3 (0.5 nM) was incubated with ADAMTS5-2 (0.5 nM) and various concentrations of PPS fractions F1–F6 (see Table 1) for 1 h at 37°C. The residual activity against Abz-TESE~SRGAIY-Dpa-KK was measured. (B) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with TIMP-3. Bound TIMP-3 was quantified using an M2 anti-FLAG antibody. TIMP-3 bound strongly to F1 (●, 35 dp), F2 (★, 25 dp), F3 (▲, 16 dp) and F4 (×, 11 dp), but only weakly to F5 (■, 8 dp) and F6 (▼, 8 dp). (C) PPS fractions F1–F6 (2.5 μM) were immobilized on glycosaminoglyan-binding multi-well plates and wells were then incubated with ADAMTS5-2. Bound ADAMTS5-2 was quantified using an M2 anti-FLAG antibody. ADAMTS5-2 bound strongly to F1 (●), F2 (★), F3 (▲) and F4 (×), and weakly to F5 (■) and F6 (▼).
Fig 3: KAT5 induces circSMARCA5 production. (A) The KAT5 expression pattern in RWPE-1, DU145, LNCaP, and PC3 cells. (B) Detection of pulled-down circSMARCA5 by qRT-PCR. (C) qRT-PCR of KAT5, miR-181b-5p, TIMP3, and miR-17-3p in DU145 cells with and without KAT5 overexpression. (D) U145 cells were transfected with circSMARCA5-expressing vector and KAT5-specific siRNA, alone or in combination. Migration and invasion were determined by Transwell assays. *, **, *** represent p ≤ 0.05, p ≤ 0.01, p ≤ 0.001, respectively. Assays were performed at least three times.
Fig 4: The schematic model of HMGA1 involved in tumor growth and metastasis of cervical cancer.First, HMGA1 accelerates the transition of G1 into S phase in cervical cancer cells by regulating cyclin D1 and cyclin E1, which further promotes tumor growth. Second, HMGA1 promotes cell migration and invasion in cervical cancer by the miR-221/222-TIMP3-MMP2/MMP9 pathway not by the miR-221/222-MMP2/MMP9 pathway, which consequently induces tumor metastasis. Lastly, HMGA1 can directly regulate the transcription of MMP2 and MMP9
Fig 5: mRNA expression levels of MMPs and associated factors in NIP and healthy control tissue. (A–F) mRNA levels of MMP-1, MMP-2, MMP-7, MMP-9, TIMP-1, TIMP-3 were detected by qRT-PCR, and analyzed with GraphPad 8. Data were shown as mean ± SD and statistical significance was analyzed using student’s t-test, *p< 0.05, ****p<0.0001.
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