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1.
Figure 1

Figure 1. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

C/EBPα-p30 expression is correlated with increased miR-181a expression. (A) Overall Survival of younger (18-59 years) CN-AML patients with CEBPA wild-type, single mutations (N or C-terminal), or double mutations (N and C-terminal mutations together). (B) miR-181a expression in younger (18-59 years) CN-AML patients with either CEBPA wild-type (CEBPA wt), monoallelic C-terminal mutations (C-term. mut.), monoallelic N-terminal mutations (N-term. mut.), or concurrent N and C-terminal mutations (N+C term. mut.). (C) Representative patient bone marrow samples with characterized CEBPA mutations effecting either the N-terminus or C-terminus of the C/EBPα protein. Quantitative real-time RT-PCR data showing the association of the CEBPA mutation status and expression of the precursor (pre) miR-181a-1 (encoded on chromosome 1 locus) or miR-181a-2 (encoded on chromosome 9 locus). Each bar represents the average of triplicate measurements and error bars denote SEM.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.
2.
Figure 3

Figure 3. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Truncated C/EBPα-p30 isoform induces expression of pre-miR-181a-1. (A) K562 cells were stably transfected with β-estradiol inducible C/EBPα-p30 or p42 ER fusion constructs31,37 enabling nuclear translocation of ectopically expressed C/EBPα proteins. For negative control, vector expressing the ER domain alone was included. Total RNA was analyzed for the expression of miR-181a-1 (black bars) and miR-181a-2 (white bars) precursors by quantitative real-time PCR. (B) Total protein lysates from K562 stable lines described in panel A were analyzed for the relative expression of C/EBPα-ER fusion proteins by Western blot stained with C/EBPα antibody. To control for loading, staining with β-tubulin antibody was used. (C) Quantitative real-time RT-PCR data and (D) Northern blot data of THP-1 cells stably expressing HA-tagged C/EBPα-p30 isoforms or empty vector.35 Mature miR-181a expression was found to be highest in those THP-1 cells expressing the HA-tagged C/EBPα-p30 isoform. Northern blot shows an increase for both pre-miR-181a-1 and mature miR-181a expression for those cells expressing the HA-tagged C/EBPα-p30 (snRNA U6 was used as an internal loading control). (E) Western blot data showing the expression of HA-tagged C/EBPα-p30 and empty vector for those cells described in panels C and D. In panels A and C, average of triplicate measurements is shown and error bars denote SEM.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.
3.
Figure 6

Figure 6. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Lenalidomide induced miR-181a expression sensitizes leukemia cells to conventional chemotherapy. (A) Untransfected THP-1 cells were cultured for 6 days in the presence of 3μM lenalidomide alone, 1μM ara-C alone, both drugs together (as indicated by “+” or “−” below the graph), or vehicle control (PBS; the left-most bar) and MTS proliferation assay was performed. The bars represent averages from 3 to 4 readings. SEM and relative percentages of proliferation rate are shown. (B) Quantitative real-time RT-PCR analysis for the expression of miR-181a in THP-1 cells transiently transfected with nontargeting control (negative control) or antagomiR-181a. Endogenous expression of miR-181a was found to be lower in those cells transfected with antagomiR-181a before drug treatments (black bars). After the treatment with 3.0μM lenalidomide, miR-181a expression was increased among those cells previously transfected with nontargeting control (red bars). In contrast, the expression of miR-181a was relatively unchanged among those cells transfected with antagomiR-181a followed by 3.0μM lenalidomide treatment (blue bars). Data are shown as an average of measurements and error bars denote SEM. (C) Western blot for those cells described in panel A with the additional treatment with either 3.0μM lenalidomide or vehicle control for 3 days. The expression of C/EBPα (p42 and p30) was found to be higher in those cells treated with lenalidomide, regardless of earlier transfection status described in panel A. Actin served as an internal loading control. (D) THP-1 cells transfected with nontargeting control, or antagomiR-181a were cultured with various concentrations (0-5μM) of cytarabine (Ara-C) in the presence of 3.0μM lenalidomide (lenalid.; solid lines), or vehicle (broken lines) for 72 hours and cellular proliferation was measured by MTS assay. Each datapoint represents an average of 3 measurements.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.
4.
Figure 7

Figure 7. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Lenalidomide treatment in vivo induces miR-181a-mediated inhibition of xenograft AML tumor growth. (A) THP-1 cells were transiently transfected with empty expression vector (control), or construct expressing the ectopic miR-181a and injected subcutaneously into NOD/SCID mice (5 mice per construct). Six weeks later the tumors were excised (left) and their sizes were determined (right). (B-E) THP-1 cells were xenografted subcutaneously to NOD/SCID mice (1 tumor per mouse). Four weeks later, tumors were directly injected with lenalidomide (lenalid.; 50 mg/kg; n = 5) or vehicle control (n = 3), twice a week for 2 weeks. Six weeks after transplantation mice were killed and tumors excised. (B) Xenograft tumors assessed at the onset (white bars) and the end (black bars) of the treatment. Measurements were plotted as the relative percentages of the tumors at the beginning of the treatment. (C) Dissected tumors after the lenalidomide treatment (lenalid.; on the right) or vehicle control (on the left). (D) Quantitative real-time RT-PCR assessment of miR-181a expression for the xenografts after treatment with either lenalidomide (lenalid.; black bar) or vehicle (white bar). (E) The relative expression of C/EBPα-p42 and C/EBPα-p30 in lenalidomide (lenalid.) or vehicle-treated xenograft tumors was evaluated by Western blot of nuclear extracts prepared from the tumors. Blot was stained with C/EBPα antibody and staining with Ku70 served as a loading control. Both lanes were from the same blot and same exposure. Vertical line has been inserted to indicate repositioned gel lanes.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.
5.
Figure 4

Figure 4. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Human miR-181a-1 promoter is regulated by C/EBPα. (A) C/EBPα binds specifically to a site within the human miR-181a-1 proximal promoter. Nuclear extracts from HEK-293T cells transiently transfected with pcDNA3-FLAG (vect.), pcDNA3-C/EBPα-p30-FLAG (α-p30), or pcDNA3-C/EBPα-p42-FLAG (α-p42) were used in electrophoretic mobility shift assay (EMSA). The radiolabeled probes contained either wild-type predicted C/EBP-binding site, or mutant (mut.) sequences (shown below). Lane labeled “probe” contains binding reaction in the absence of nuclear extract. Where indicated by “+” above the lanes, C/EBPα specific antibody was added to the binding reactions. Solid arrowhead shows protein/DNA complex, whereas open arrowhead indicates binding complex super-shifted with the antibody. Unbound probe is shown on the bottom of the gel (free probe). (B) Relative expression of C/EBPα proteins in nuclear extracts used in EMSA in panel A is demonstrated in Western blot stained with C/EBPα-specific antibody. The p42 and p30 C/EBPα polypeptides are indicated to the right. (C) C/EBPα-dependent transactivation of the miR-181a-1 promoter. Human miR-181a-1 192 bp promoter fragment containing wild-type C/EBP-binding site (boxed sequence) or mutated sequence (indicated below the box) were linked to firefly luciferase gene (black box; diagrammed on top) and transiently transfected to HEK-293T cells with either empty expression vector pcDNA3-FLAG (−), or pcDNA3-C/EBPα-FLAG vectors (p42 or p30). Cell lysates were analyzed for luciferase activity and normalized to cotransfected Renilla luciferase activity. For control, C/EBPα expression vectors were also tested with promoter-less luciferase vector, pGL4-11 (vector). Each bar represents average of 3 transfection experiments and SEM bars are shown. On the right, representative aliquots of cell lysates used for luciferase assay were also analyzed by Western blot to demonstrate comparable levels of C/EBPα protein.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.
6.
Figure 5

Figure 5. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

Increased C/EBPα-p30 expression after treatment with the immunomodulatory compound, lenalidomide. (A) Western blot analysis of AML patient blasts treated in vitro with 3.0μM lenalidomide followed by hourly collections. The expression of C/EBPα (p30 and p42) was detected by C/EBPα antibody. The signal intensities were assessed and the p30/p42 ratios were calculated (shown below C/EBPα stained blot). Staining with Actin antibody served as an internal loading control. The data shown are representative for 3 patient samples. (B) Quantitative real-time RT-PCR data for miR-181a expression in the same AML blasts shown in panel A. The expression of miR-181a was increased at 12 hours and 24 hours after 3.0μM lenalidomide treatment (black bars) compared with the vehicle control (white bars) for the same time point. Average of triplicate measurements from a single patient sample is shown and error bars denote SEM. Similar results shown in panels A and B were observed in a total of 3 separate AML patient blasts used in the same experiment (not shown). Additional data from the long-term in vitro treatment with lenalidomide are shown in supplemental Figure 3. (C) Quantitative real-time RT-PCR data from 3 bone marrow samples from patients treated with lenalidomide induction therapy (top). Samples were analyzed before treatment (white bars) and 5 days after lenalidomide induction therapy (black bars). Data are shown as average of triplicate measurements and error bars denote SEM. Increased expression of miR-181a was observed on day 5 after lenalidomide induction therapy. Corresponding whole cell lysates for those patients were analyzed using Western blot (bottom). The expression of C/EBPα (p42 and p30) was increased on day 5 after lenalidomide induction therapy. Actin served as an internal loading control. Patients' cytogenetic and clinical characteristics before the therapy are summarized in supplemental Table 1.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.
7.
Figure 2

Figure 2. From: Lenalidomide-mediated enhanced translation of C/EBP?-p30 protein up-regulates expression of the antileukemic microRNA-181a in acute myeloid leukemia.

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.

Christopher J. Hickey, et al. Blood. 2013 January 3;121(1):159-169.

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