Fig. 4.

CircAF4 acts as a miR-128-3p sponge, releasing its inhibition on MLL-AF4 expression. a Western blotting confirmation of the effects of forced expression and knockdown of circAF4 by lentiviral constructs on MLL-AF4 protein level in RS4;11 cells. The MLL-N320 or -AF4/ Tubulin densitometric ratio was recorded by ImageJ. b Schematic representation of the constructs used in the luciferase assay. The sequences shown below indicate the putative miR-128-3p target site on the wild-type 3′ UTR (construct circAF4-wt), its mutated derivative (construct circAF4-mut), and the pairing regions of miR-128-3p. Luciferase assays show that transfection miR-128-3p mimic repressed the reporter activity of circAF4-wt, but not circAF4-mut. NS, not significant. ***P < 0.001. c Luciferase assays show that transfection miR-128-3p mimic repressed the reporter activity of MLL-AF4-3′UTR, and such repression could be partially restored by overexpression of circAF4. *P < 0.05 and **P < 0.01. d RNA immunoprecipitation (RIP) of AGO2 in RS4;11 and MV4-11 cells using the Magna RIP RNA-Binding Protein Immunoprecipitation Kit (Millipore, Bedford, MA) according to the manufacturer’s instructions. Analysis of precipitated RNAs: circAF4 and U6, negative control. Data are given as mean ± SEM. e Co-localization between miR-128-3p and circAF4 was observed by RNA in situ hybridization in RS4;11 cells. CircAF4 oligonucleotides were labeled with rhodamine (red), and miR-128-3p oligonucleotides were labeled with FAM (green). Nuclei were stained with DAPI (blue). Scar bars, 5 μm. f Flow cytometric analysis of Annexin V and PI positive cells in RS4;11 and MV4-11 cells transfected with indicated siRNA or miRNA mimics, respectively. *P < 0.05, **P < 0.01, and ***P < 0.001