Fig. 2

RNA binding protein FUS increases the stability of circAAGAB via direct binding. A Venn diagram of the predicted RNA binding proteins (RBPs). RNA binding proteins with circAAGAB were predicted by ENCORI (http://starbase.sysu.edu.cn/) and RBPDB (http://rbpdb.ccbr.utoronto.ca/). * The 4 common RBPs are shown. B The predicted possibility of interaction between circAAGAB and RBP. The binding possibility was calculated by RNA–Protein Interaction Prediction (RPISeq) (http://pridb.gdcb.iastate.edu/RPISeq/) using random forest (RF) and support vector machine (SVM) classifiers. Predictions with probabilities > 0.5 were considered positive. C RNA pull-down assay. Cell lysates were extracted from MDA-MB-231 cells, pulled down by biotin-labeled probe against circAAGAB, and subjected to western blotting using FUS antibody. D Expression levels of RNA in MDA-MB-231 cells transfected with siRNA against FUS by quantitative RT-PCR. NC: nontargeting control siRNA. Internal control: GAPDH. E Stability assay of circAAGAB. MDA-MB-231 cells were transfected with 5 μg/mL actinomycin D and siRNA against FUS. The expression levels of circAAGAB were measured by quantitative RT-PCR at various time points. NC: nontargeting control siRNA. Internal control: GAPDH. *P < 0.05, **P < 0.01