Fig. 3

Tspan7 contributes to OS cell metastasis in vitro. A Cells in which Tspan7 was stably knocked down using the indicated shRNA constructs exhibited reduced Tspan7 mRNA levels and protein levels as compared to the shNC group in qRT-PCR (left panel) and western blotting (right panel) assays. B Cells were imaged at 10× magnification, with successfully infected cells exhibiting GFP expression, demonstrating near 100% transduction efficiency. C, D The effect of Tspan7 downregulation on HOS and Saos2 cell migration was assessed by wound healing and Transwell assays. E Matrigel-coated invasion assays were used to assess the invasivity of HOS and Saos2 cells stably expressing shTspan7 or shNC. F Fluorescence microscopy (upper panel), qRT-PCR (lower left panel), and western blotting (lower right panel) were used to gauge the degree of Tspan7 overexpression in OE-Tspan7 and mock U2OS cells. G Wound healing assays were conducted using U2OS cells stably transfected with OE-Tspan7 or mock constructs. H U2OS cells stably transfected with OE-Tspan7 or mock constructs were utilized in Transwell migration assays. I Matrigel-coated invasion assays were used to assess the invasivity of U2OS cells stably expressing OE-Tspan7 or mock constructs. Representative images are shown, and all migratory and invasive cells were counted. Experiments were repeated in triplicate, and data are means ± SD. shRNA, short hairpin RNA; GFP green fluorescence protein. *P < 0.05, **P < 0.01, ***P < 0.001, ****P < 0.0001. Student’s t-test