Fig. 6

TXNIP negatively regulates glucose metabolism and proliferation in osteosarcoma. a. Overexpression of CircECE1 in OS cells, and heat map detailing the RNA-seq. b. Knockdown of c-Myc or CircECE1 increased the levels of TXNIP mRNA. c. Luciferase assay showed that c-Myc binds to the TXNIP promoter. d. c-Myc occupies the E-box of the TXNIP promoter region as measured by ChIP assay. e. Knockdown of c-Myc increased TXNIP protein expression. f. Effect of TXNIP overexpression or knockdown on the expression levels of genes involved in glucose metabolism in OS cells as assessed by real-time PCR. g. TXNIP inhibited cell proliferation as measured by a CCK-8 proliferation kit. h. TXNIP inhibited cell proliferation as measured by a colony formation assay. i. Cell lysates were collected, and immunoblotting was performed using the indicated antibodies. j. TXNIP rescued the expression levels of genes involved in glucose metabolism in OS cells by real-time PCR, and this was induced by CircECE1 overexpression. k-m. ATP and lactate production was lower in the TXNIP and CircECE1 co-expressing OS cells. n. TXNIP and CircECE1 co-expression lowered the glycolysis rate as indicated by the ECAR and OCR. o. TXNIP and CircECE1 function in tumor cell proliferation as detected by CCK-8 assay. p. TXNIP and CircECE1 function in tumor cell proliferation as detected by EdU assay. Nuclei were stained with DAPI. A combined reaction between EdU and DAPI identified the cells in S phase. q. Cell migration abilities of U2OS and 143B cells transfected with CircECE1 and TXNIP, CircECE1 alone, or vector were evaluated using transwell migration assays. Data represent the mean ± SD (n = 3). * P < 0.05. Scale bar = 50 μm. r. TXNIP and CircECE1 overexpression in osteosarcoma cells increased cell anoikis (n = 3) compared to that observed in response to CircECE1 alone