American Society of Hematology

Figure 2

$Figure 2. Forced expression of patient-derived N-terminal mutated CEBPA into K562 cells induces up-regulation of the endogenous miR-181a. (A) Schematic diagram of C/EBPα expressed from vectors containing the CEBPA gene isolated from AML patients with wild-type CEBPA or identified CEBPA mutations. N-terminal mutations locations, P23Qfs and H24Afs, are depicted by inverted triangles on the wild-type protein. N-terminal mutations produce the N-truncated C/EBPα-p30 isoform. C-terminal mutation contains an amino acid substitution R300L located within the nuclear localization sequence domain (V-K; underlined amino acids are believed to be required for nuclear localization). DNA-binding domain (DBD) and leucine-zipper (L-ZIP) domain are represented by white and black boxes, respectively. (B) Confocal microscopy images for K562 cells transiently expressing those C/EBPα isoforms described in panel A. All C/EBPα isoforms are identified with green labeling and DAPI staining performed as described,50 indicates location of nucleus shown in blue. The wild-type C/EBPα and N-terminal mutant C/EBPα were localized to the nucleus. In contrast, the C-terminal mutant C/EBPα (R300L) was localized in the cytoplasm. (C) Western blot analysis of cellular fractions after ectopic expression of C/EBPα isoforms described in panel A and expressed in the leukemia cell line K562. Notably, all isoforms are localized in the nucleus except for R300L which is predominately cytoplasmic. UBC9 serves as a positive-control for C/EBPα-p30 expression.33 Ku-70 and Actin serve as internal loading controls. (D) Quantitative real-time RT-PCR analysis for miR-181a expression in the K562 transiently transfected with the expression constructs described in panel A. Notably, miR-181-1 expression is highest in those cells transiently expressing the C/EBPα-p30 isoform. Each bar represents average of triplicate measurements and error bars denote SEM. (E) FAM fluorescence images of KG1a cells transfected with C/EBPα expression plasmids (N-terminal or C-terminal mutants, wild-type C/EBPA, or empty vector) and 1 day later with a fluorescent miR-181a LNA-MB (left), assessed for relative expression of endogenous miR-181a levels (right). FAM is released from its quencher and emitted fluorescence when MBs unfold and bind to miR-181a. Average of 3 to 9 (n) measurements is shown and error bars denote SEM.$

Forced expression of patient-derived N-terminal mutated CEBPA into K562 cells induces up-regulation of the endogenous miR-181a. (A) Schematic diagram of C/EBPα expressed from vectors containing the CEBPA gene isolated from AML patients with wild-type CEBPA or identified CEBPA mutations. N-terminal mutations locations, P23Qfs and H24Afs, are depicted by inverted triangles on the wild-type protein. N-terminal mutations produce the N-truncated C/EBPα-p30 isoform. C-terminal mutation contains an amino acid substitution R300L located within the nuclear localization sequence domain (V-K; underlined amino acids are believed to be required for nuclear localization). DNA-binding domain (DBD) and leucine-zipper (L-ZIP) domain are represented by white and black boxes, respectively. (B) Confocal microscopy images for K562 cells transiently expressing those C/EBPα isoforms described in panel A. All C/EBPα isoforms are identified with green labeling and DAPI staining performed as described,⁵⁰ indicates location of nucleus shown in blue. The wild-type C/EBPα and N-terminal mutant C/EBPα were localized to the nucleus. In contrast, the C-terminal mutant C/EBPα (R300L) was localized in the cytoplasm. (C) Western blot analysis of cellular fractions after ectopic expression of C/EBPα isoforms described in panel A and expressed in the leukemia cell line K562. Notably, all isoforms are localized in the nucleus except for R300L which is predominately cytoplasmic. UBC9 serves as a positive-control for C/EBPα-p30 expression.³³ Ku-70 and Actin serve as internal loading controls. (D) Quantitative real-time RT-PCR analysis for miR-181a expression in the K562 transiently transfected with the expression constructs described in panel A. Notably, miR-181-1 expression is highest in those cells transiently expressing the C/EBPα-p30 isoform. Each bar represents average of triplicate measurements and error bars denote SEM. (E) FAM fluorescence images of KG1a cells transfected with C/EBPα expression plasmids (N-terminal or C-terminal mutants, wild-type C/EBPA, or empty vector) and 1 day later with a fluorescent miR-181a LNA-MB (left), assessed for relative expression of endogenous miR-181a levels (right). FAM is released from its quencher and emitted fluorescence when MBs unfold and bind to miR-181a. Average of 3 to 9 (n) measurements is shown and error bars denote SEM.

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