Functional significance of circRNA expression in AML. (A-B) Relative expression of linPCMTD1, circPCMTD1, linKLHL8, circKLHL8, linCFLAR, circCFLAR, linFBXW7, and circFBXW7 in KG-1a (A) and OCI-AML3 (B) cells. (C-D) Circular-to-linear (circ/lin) ratio of the PCMTD1, KLHL8, CFLAR, and FBXW7 transcripts in mock vs RNase R–treated samples of KG1a (C) and OCI-AML3 (D) cells. The log2 value of the circ/lin ratio is depicted in the figures. (E) Schematic description of the PCR experiments with divergent primers. The reverse primer (primer A) binds to the 5′ end of exon 1 and the forward primer (primer B) binds to the 3′ end of exon 2. With this design, amplicons can be generated after hybridization of the primers on circular RNA transcripts but not on genomic DNA. (F) circPCMTD1, circKLHL8, circCFLAR, and circFBXW7 PCR results using complementary DNA and genomic DNA of KG-1a cells. The integrity of the DNA was tested with primers specific for the ACTB DNA promoter region. (G-H) Proliferation analyses of scramble vs circFBXW7 KD-treated KG-1a (G) and OCI-AML3 (H) cells, as measured by a colorimetric assay, based on degradation of the WST1 reagent. (I-J) Proliferation analyses of scramble vs circFBXW7 KD-treated KG-1a (I) and OCI-AML3 (J) cells, based on BrdU incorporation. (K-M) Relative RNA expression of linFBXW7 and circFBXW7 in scramble vs circFBXW7 KD-treated KG-1a cells (K), OCI-AML3 cells (L), and AML patient blasts (M). (M) Samples of 1 patient are depicted as an example. (N) Colony-forming unit assays in methylcellulose-based media with scramble vs circFBXW7 KD-treated AML patient blasts of 3 different patients. N.S., not significant.