Fig 2: Structure of ALS and key determinants of IGF1/IGFBP3 binary complex.a Two different views of the horseshoe-like structure of ALS. LRRNT (yellow), LRR 1–19 (violet), and LRRCT (lime) of ALS are labeled. The conserved asparagine-ladder (blue) and phenylalanine-spine (red) are shown as sticks. Descending and ascending flanks of ALS are labeled (bottom). b Close-up view of the ALS hook loop (boxed in A). Residues involved in stabilization of the ALS hook loop are displayed as sticks and labeled. a, b The glycosylation sites (N64, N96, N368, N515, and N580) and disulfide bridges (C41–C47 and C45–C60 in LRRNT, C373–C397 in LRR motif, C540–C583, C542–C605, and C566–C571 in LRRCT) are indicated. Disulfide bridges (orange) and N-linked glycans (green) are shown as sticks. c Comparison of the IGF1/IGFBP3 in the ternary complex with the structure of IGF1/NBP4/CBP4 complex (gray, PDB: 2DSR). Structures are shown as ribbon diagrams. d–f Close-up views of the interacting interface between the IGF1 B domain ? helix and the cleft formed by NBP3 and CBP3 (d), interaction at the interface between IGF1 and the entrance of the cleft, and direct contact between NBP3 and CBP3 (e), and ß sheet formed by IGF1, CBP3, and the N-terminal thumb of NBP3 (f). The view for e is a rotation of the view for d ~150° about the y-axis. Key residues involved in each interaction are displayed as sticks and labeled. The ? helix (A8-V17) and ß strand (G22-Y24) of the IGF1 B domain and the ?1 helix (I43-F49) and ?2 helix (L54-Y60) of the IGF1 A domain are labeled as ?B, ßB, ?A1, and ?A2, respectively.

Fig 3: Critical role of CLD3 for sequential assembly of IGF1/IGFBP3/ALS ternary complex.a SDS-PAGE analysis of the co-expression test. ALS-His, IGFBP3-Strep, and IGF1 (IGF1 or IGF1-Strep) are co-expressed and pulled down with Ni-NTA or Strep-Tactin resin, as indicated. b Co-expression of CLD3-CBP3 with ALS and/or IGF1. ALS-His, CLD3-CBP3-Strep, and IGF1 (IGF1 or IGF1-His) were co-expressed and pulled down with Ni-NTA or Strep-Tactin resin, as indicated. c Fluorescence-detection size-exclusion chromatography (FSEC) after in vitro reconstitution of indicated proteins. Fluorescence signal (GFP) fused to IGF1 was monitored to examine the complex formation. The elution profiles for standard IGF1-GFP (yellow), IGF1-GFP/IGFBP3 binary complex (blue), IGF1-GFP/IGFBP3/ALS ternary complex (red), and ALS alone (purple) are indicated as a control (top). d Co-expression of hybrid-BPs with ALS and IGF1. Schematic diagram of the human IGFBP3 and hybrid-BPs (top). Each domain is labeled above the diagram. ALS-His and IGF1 were co-expressed with IGFBP3 (lane 1) or hybrid-BPs (lanes 2–6) and pulled with Ni-NTA resin, then analyzed by SDS-PAGE. e Co-expression of hybrid-BPs with ALS. ALS-His were co-expressed with IGFBP3 (lane 1) or hybrid-BPs (lanes 2–6) and pulled with Ni-NTA resin, then analyzed by SDS-PAGE. a, b, d, e Similar results were observed in three independent experiments.

Fig 4: Circ_0000285/miR-409-3p/IGFBP3 axis was associated with OS progression. a–e Cell viability, migration, invasion, colony numbers, cycle and apoptosis were detected in si-NC, si-circ_0000285, si-circ_0000285 + inhibitor NC and si-circ_0000285 + miR-409-3p inhibitor groups. f–j Cell viability, migration, invasion, colony numbers, cycle and apoptosis were detected in miRNA NC, miR-409-3p mimic, miR-409-3p mimic + pc-NC and miR-409-3p mimic + pc-IGFBP3 groups. *P < 0.05

Fig 5: Circ_0000285 sponged miR-409-3p to target IGFBP3 in OS. a qRT-PCR was used to detect the expression of miR-32-3p, miR-548-5p, miR-127-5p, miR-1278, miR-2467-3p, miR-4731-5p, miR-599 and miR-409-3p in si-NC and si-circ_0000285 groups in SJSA1 and U2OS cells. b The predicted binding sites of miR-409-3p to the circ_0000285 and IGFBP3 sequences were shown. c, d Luciferase activities were detected in SJSA1 and U2OS cells co-transfected miR-409-3p mimic or miRNA NC with WT-circ_0000285 or MUT-circ_0000285 and WT-IGFBP3-3′UTR or MUT-IGFBP3-3′UTR. e, f RIP assay was applied to determine the relationship among circ_0000285, miR-409-3p and IGFBP3. *P < 0.05, **P < 0.01, ***P < 0.001