Figure 3.

circ-Foxo3 interacted with CDK2 and P21. (A) Cell lysis prepared from NIH3T3 cells were subject to immuno-precipitation (IP) with antibodies against rabbit IgG, mouse IgG, cyclin A, cyclin B, cyclin C, cyclin D1, cyclin E, CDK2, CDK4, CDK6, p16, p18, p21, p27 and p57, followed by real-time PCR with primers specific for linear Foxo3 mRNA or circ-Foxo3. Anti-CDK2, CDK6, p16, p21and p27 antibodies pulled-down circ-Foxo3, but not linear Foxo3 mRNA. It was especially obvious that precipitating CDK2 or P21 pulled-down circ-Foxo3. **P < 0.01. Error bars, SD (n = 4). (B) Cell lysates prepared from NIH3T3 cells transfected with circ-Foxo3 or mock control were subject to immunoprecipitation with anti-rabbit IgG, mouse IgG, cyclin A, cyclin B, cyclin C, cyclin D, cyclin E, CDK2, CDK4, CDK6, p16, p18, p21, p27 and p57 antibodies. Western blot showed that immunoprecipitation pulled down similar amount of proteins in both control and circ-Foxo3-transfected cells. (C) The immuno-precipitated mixtures were also subject to real-time PCR with primers specific for circ-Foxo3. Anti-CDK2, CDK6, p16, p21and p27 antibodies pulled-down more circ-Foxo3 from NIH3T3 cells transfected with circ-Foxo3 than from mock control. *P < 0.01. Error bars, SD (n = 4). (D) NIH3T3 cells were subject to flow cytometry to sort cells in G1, S and G2 phases, followed by immunoprecipitation with anti-rabbit IgG, mouse IgG, p21 and Cdk2 antibodies and real-time PCR with primers specific for circ-Foxo3. Antibodies against p21 and Cdk2 precipitated significantly more circ-Foxo3 in G1 phase than in G2 an S phases. **P < 0.01. Error bars, SD (n = 4). (E) Cell lysates prepared were subject to immunoprecipitation with anti-rabbit IgG, mouse IgG, p21 and Cdk2 antibodies, followed by real-time PCR with primers specific for circ-Foxo3, circ-DNAJA1, circ-MRPL47, circ-NDUF53, circ-RPS5 and circ-RPF5. Antibodies against p21 and Cdk2 pulled-down circ-Foxo3, but not the other circular RNAs. **P < 0.01. Error bars, SD (n = 4).