Figure 1
Figure 1. C/EBPα-p42 regulates miR-34a during granulopoiesis. (A-C) Hematopoietic CD34+ cells were cultured as discussed in “Cell cultures.” Total RNA was isolated at different time points and analyzed by quantitative real-time RT-PCR with oligos for miR-34a (A), miR-223 (B), and miR-181a (C). Data are represented as mean ± SD from 3 independent experiments. *P < .05. (D) NB4 cells were induced with retinoic acid (1μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-34a. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (E) K562-C/EBPα-p42-ER and K562-ER cells were induced with β-estradiol (5μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-34a. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (F) K562-C/EBPα-p30-ER and K562-C/EBPα-BRM2-ER cells were induced with β-estradiol (5μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-34a. Data are represented as mean ± SD from 3 independent experiments. (G) Schematic representation of the miR-34a genomic region and phylogenic conservation of genomic region in the first intron of miR-34a in humans, mice, and rats. The conserved region is shown by sequences in gray box, and the C/EBPα site is shown in bold letters. (H) Luciferase reporter assays were performed in Kasumi-6 cells using indicated reporters and C/EBPα. Cells were transfected with corresponding firefly luciferase vectors (miR-34 Promoter constructs #1 to #6, Renilla luciferase reporter construct as control vector, and C/EBPα-pCDNA3 vector. Luciferase activity was measured 24 hours later. Bars represent promoter activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (I) Chromatin derived from K562-C/EBPα-p42-ER cells was immunoprecipitated with anti-C/EBPα and IgG antibodies. Recovered DNA was PCR amplified with primers specific for C/EBPα-binding amplicon (oligos 1) and the nonbinding amplicon (oligos 2).

C/EBPα-p42 regulates miR-34a during granulopoiesis. (A-C) Hematopoietic CD34+ cells were cultured as discussed in “Cell cultures.” Total RNA was isolated at different time points and analyzed by quantitative real-time RT-PCR with oligos for miR-34a (A), miR-223 (B), and miR-181a (C). Data are represented as mean ± SD from 3 independent experiments. *P < .05. (D) NB4 cells were induced with retinoic acid (1μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-34a. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (E) K562-C/EBPα-p42-ER and K562-ER cells were induced with β-estradiol (5μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-34a. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (F) K562-C/EBPα-p30-ER and K562-C/EBPα-BRM2-ER cells were induced with β-estradiol (5μM) for respective time points. Total RNA was analyzed by quantitative real-time RT-PCR with oligos for miR-34a. Data are represented as mean ± SD from 3 independent experiments. (G) Schematic representation of the miR-34a genomic region and phylogenic conservation of genomic region in the first intron of miR-34a in humans, mice, and rats. The conserved region is shown by sequences in gray box, and the C/EBPα site is shown in bold letters. (H) Luciferase reporter assays were performed in Kasumi-6 cells using indicated reporters and C/EBPα. Cells were transfected with corresponding firefly luciferase vectors (miR-34 Promoter constructs #1 to #6, Renilla luciferase reporter construct as control vector, and C/EBPα-pCDNA3 vector. Luciferase activity was measured 24 hours later. Bars represent promoter activity for the corresponding vectors. Data are represented as mean ± SD from 3 independent experiments. *P < .05. (I) Chromatin derived from K562-C/EBPα-p42-ER cells was immunoprecipitated with anti-C/EBPα and IgG antibodies. Recovered DNA was PCR amplified with primers specific for C/EBPα-binding amplicon (oligos 1) and the nonbinding amplicon (oligos 2).

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