Fig. 2

Tumor suppressive functions of circKEAP1 in vivo and in vitro. (A). OS cells were transfected with circKEAP1, two circKEAP1 shRNAs or control vectors. Colony formation of OS cells was determined in the indicated transfection groups. (B). Caspase 3 and PARP levels were determined by western blotting in the indicated transfection groups. (C). Apoptosis of the indicated OS cells was detected by flow cytometry. (D). Cell migration of the indicated OS cells was determined by wound healing assay. (E). Cell migration of indicated OS cells was determined by transwell assay. (F). The indicated U2OS cells (NC and circKEAP1) were injected into nude mice via tail vein and the lungs were harvested after 35 days. Representative image and H&E staining of mouse lung metastasis is shown (scale bars, 200 μm). (G). The indicated U2OS cells (NC and circKEAP1) were injected subcutaneously into nude mice (n = 5 per group) and tumor volume was measured every 7 days. After 28 days, tumors were collected and weighed. (H). Tumor tissues stained with Ki67 antibody and IHC score are shown (scale bars, 50 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