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Figure 4 Trio knockdown in NCCs negatively affects cell differentiation and migration in vitro. (A) Wound healing assays conducted and photographed at 0, 12, and 24 h, with quantification (n = 5). Bar = 200 ?m. (B and C) Transwell migration assay of NCCs at 24 and 36 h after Trio knockdown, with quantification (n = 5). Bar = 100 ?m. (D) Immunostaining of F-actin (red) in the cytoskeleton of shCtrl and shTrio NCCs, with quantification (n = 5). Nuclei were counterstained with DAPI (blue). Bar = 100 ?m. DAPI: 4, 6-diamidino-2-phenylindole. (E) Immunostaining for ?-tubulin (green) in the cytoskeleton of shCtrl and shTrio NCCs. The nuclei were counterstained with DAPI (blue). Bar = 100 ?m. (F) Osteogenesis assessment using alkaline phosphatase (ALP) staining with quantification of NCC differentiation after five days of incubation in osteogenic medium (n = 8). Bar = 100 ?m. shCtrl: short hairpin control (control lentivirus); shTrio: short hairpin Trio (Trio lentivirus). (G) Osteogenic differentiation assessment using Alizarin red staining (ARS) in NCCs after 14 days of incubation in osteogenic medium, with quantification (n = 8). Bar = 100 ?m. (H) CCK8 assay at 0, 1, 3, 5, 7 days (n = 5). (I) Cell fractions of different phases detected by cytometry (n = 3). (J) Cell apoptosis measured by cytometry, with quantification analysis (n = 3). For (A) - (D) and (F) - (J), data are represented mean ± S.E.M. (two-tailed t test *p < 0.05, **p < 0.01, ns, not significant).