Fig. 2

CircECE1 increased cell survival and colony formation and promoted the expression of c-Myc targets. a. Silencing or overexpression of CircECE1 in osteosarcoma cells decreased or increased, respectively, the ability of cell proliferation compared to that of the control. **P < 0.01. Data represent the mean ± SD (n = 6). b. CircECE1 functions in tumor cell proliferation as detected by EdU assay. Nuclei were stained with DAPI, and a combined reaction involving EdU and DAPI indicated the cells in S phase. c. CircECE1 overexpression promotes cell growth as determined by colony formation assay (details are shown in the insets). Error bars represent the mean ± SD of three independent experiments. * P < 0.05. d. Cellular transformation was induced by CircECE1 overexpression. Vector or CircECE1-overexpressing OS stable cells were cultured in soft agar for 20 days. Colonies were stained with crystal violet, photographed, and quantified using ImageJ (details are shown in inserts). e. CircECE1 downregulation in osteosarcoma cells increased cell apoptosis (n = 3). f. CircECE1 overexpression in osteosarcoma cells decreased cell anoikis (n = 3). g. Cell migration abilities of U2OS and 143B cells transfected with CircECE1 or vector were evaluated by transwell migration assays. Data represent the mean ± SD (n = 3). * P < 0.05. Scale bar, 50 μm. h. Protein lysates from vector and CircECE1 or shCircECE1-transfected osteosarcoma cells were subject to western blotting. Cell lysates were analyzed using c-Myc, HIF-1α, Cdc25a, Mdm2, ELK1, JUN, and JUNB antibodies. i. The shCircECE1 or CircECE1 and the vector transfected osteosarcoma cells were maintained at 80% confluence and were used for RNA extraction and measurement of c-Myc and c-Myc targets. The CircECE1 cells expressed significantly higher levels of c-Myc targets compared to those of the control