Fig. 4

KEAP1-259aa exerts the biological function in OS cells. (A). OS cells were transfected with empty vector, circKEAP1 vector, ATG-mutated circKEAP1 vector and IRES-mutated circKEAP1 vector. Colony formation was performed in the indicated transfection groups. (B). Cell proliferation of the indicated cells was characterized by CCK-8 assay. (C). Apoptosis of the indicated cells was detected by flow cytometry. (D). Cell apoptosis of the indicated cells was characterized by western blotting assay. (E). Cell migration of the indicated cells was characterized by wound healing assay. (F). Cell migration of the indicated cells was characterized by transwell assay. (G). Tumor sphere assay was performed to detect stemness of the indicated cells (scale bar, 50 μm). (H). Flow cytometry assay was used to examine percentage of CD133 + cells in the indicated cells. (I). SOX2, ABCG2, Oct4 and Nanog expression in cells was characterized by western blotting. (J). The indicated cells (1 = NC, 2 = KEAP1-259aa, 3 = IRSE mut, 4 = ATG mut) were injected subcutaneously into nude mice (n = 4 per group) and tumor volume was measured every 7 days. After 28 days, tumors were collected and weighed. (K). The indicated cells were injected into nude mice (n = 4 per group) via tail vein and the lungs were harvested after 35 days. Representative image and H&E staining of mouse lung metastases is shown (scale bars, 200 μm). Error bars represent three independent experiments. *, **, *** indicates significant differences compared with the control group at a p value < 0.05, < 0.01, < 0.001, respectively