Figure 1.
Cebpa knockdown increases murine marrow cell monopoiesis relative to granulopoiesis and generates a blast population.
A) Diagram of marrow transduction protocol. B) Western blot for C/EBPα and β-actin after transduction and puromycin selection with the pLKO.1 vector (Vec) or with Cebpa shRNAs B9 or B11. C) Number of CFU-M, CFU-G, or CFU-GM per 1E4 cells plated in methylcellulose culture with IL-3, IL-6, and SCF (n = 6–9). D) FACS analysis for Mac-1 and Gr-1 on pooled CFUs. Arrows indicate immature, lineage-negative cells (top). Morphology of pooled CFU cells (bottom). *p<0.05, **p<0.01, ***p<0.001.
Figure 2.
CEBPA knockdown increases human marrow cell monopoiesis relative to granulopoiesis.
A) Diagram of marrow transduction protocol (top) and CEBPA mRNA expression normalized to β-Actin on D0 after transduction with non-targeting vector (NTV) or CEBPA shRNAs G5 or G6. B) FACS analysis for the CD14 (monocytic) and CD15 (granulocytic) markers at D7 for liquid cultured cells. C) Number of CFU-M, CFU-G, and CFU-GM per 5E4 cells plated (n = 3). D) Representative morphology of pooled CFU cells. Note large monocytes with abundant basophilic cytoplasm and smaller granulocytic cells with eosinophilic cytoplasm and granules. The proportion of monocytes (M) and granulocytes (G) amongst 100 cell counted are shown.
Figure 3.
Cebpa knockdown increases myeloid colony replating.
A) Myeloid CFUs obtained after transduction with the pLKO.1 vector or with Cebpa shRNAs B9 or B11 were replated each 7 days. The average number of generations for which CFUs were obtained is plotted (top, n = 4–5). G4 indicates that initial CFUs could be replated 3 times to yield CFUs. CFU-G and CFU-M obtained at each generation from a typical experiment done in triplicate, 1E4 cell/plate (bottom). B) FACS analysis for Mac-1 and Gr-1 from G3 CFUs. Arrows indicate immature, lineage-negative cells (top). Morphology of G4 and G6 CFUs from the same experiment (bottom).
Figure 4.
Cebpa knockdown reduces murine granulopoiesis relative to monopoiesis and increases immature blasts in liquid culture.
A) Transduced, puromycin selected marrow cells were placed in liquid culture with IL-3, IL-6, and SCF on D0 and viable cell counts were enumerated on days 0, 2, 3, 4, and 7. Representative data is shown. B) Cell cycle analysis was conducted using PI staining on D0 or D3. C) FACS analysis for Mac-1 and Gr-1 on D2. Arrows indicate immature, Mac-1−Gr-1− cells (top). FACS analysis of Mac-1−Gr-1− cells stained for Sca-1 and c-Kit (middle). FACS analysis of D2 cells for FcγR and CD34, gating on Lin−Sca-1−c-Kit+ progenitors (bottom). Representative data are shown. D) Total cellular RNAs collected on day 3 (D3) after lentiviral transduction with the pLKO.1 vector or with Cebpa shRNAs B9 or B11, puromycin selection, and transfer to IL-3, IL-6, and SCF were subjected to quantitative RT-PCR for indicated mRNAs (n = 3).
Figure 5.
Cebpa knockdown reduces murine granulopoiesis and increases monopoiesis and immature cells in vivo.
A) Diagram of transduction and transplantation protocol using the pLKO.1 vector or Cebpa shRNAs B9 or B11. B) Marrow cells isolated 28 days after transplantation (D28) were subjected to CD45.2/CD45.1 or Mac-1/Gr-1 FACS analysis. Representative data are shown. C) Summary of Sca-1/c-Kit and Mac-1/Gr-1 FACS analyses (vector and B9, n = 4; B11, n = 3). D) On day 28, CD45.2+CD45.1− marrow cells isolated by flow cytometry were plated in methylcellulose with IL-3, IL-6, and SCF. The number of CFU-M, CFU-G, or CFU-GM obtained per 1E4 cells plated is shown (n = 3).
Figure 6.
32Dcl3 cells with partial Cebpa knockdown retain capacity for nearly complete granulocytic maturation.
A) Parental 32Dcl3 cells, pools of 32Dcl3 cells stably transduced with the pLKO.1 vector (Vec) or Cebpa B9 or B11 shRNAs, or subclones of the B9 or B11 pools were subjected to Western blotting for C/EBPα and β-actin. B) Indicated 32Dcl3 lines were subjected to FACS analysis for GCSFR using biotin-G-CSF in the absence (heavy line) or presence (light line) of excess unbiotinylated G-CSF; x-axis is log scale (top). RNAs prepared from indicated 32Dcl3 lines cultured in IL-3 or after one or four days in G-CSF (G1, G4) were subjected to quantitative RT-PCR for Mpo (bottom, n = 3). C) Vector, B11-1, or B9-2 cells cultured in GCSF were subjected to Wright-Giemsa staining on the days of maximal differentiation, G6, G8, and G4 respectively (top row). Vector/GR, B11-1/GR, or B9-2/GR, G14, G14, and G6, respectively, were analyzed similarly (bottom row). D) Pools of vector, B11-1, or B9-2 lines transduced with pBabeNeo or pBabeNeo-GCSFR were subjected to FACS analysis for GCSFR (top). RNAs prepared from these lines in IL-3 or G-CSF were analyzed for Mpo expression (bottom, n = 3).
Figure 7.
C/EBPα-ER rescues the differentiation blocked induced in murine marrow or 32Dcl3 cells by Cebpa knockdown.
A) Diagram of marrow transduction protocol with lentiviral pLKO.1(puro)-B9 shRNA and MIG or MIG-C/EBPα-ER(B9res) retrovirus (top). 293T whole cell extracts obtained 2 days after transfection with MIG or MIG-C/EBPα-ER(B9res) DNAs were subjected to Western blotting with C/EBPα or actin antibodies (center, right). Representative Mac-1;Gr-1 FACS analysis of marrow cells 2 days after transfer to IL-3, IL-6, SCF +/− E2 (bottom, left). Average reduction in Mac-1−Gr-1−(Lin−) cells or increase in percent monocytes compared to total monocytes + granulocytes (n = 3, bottom, right). B) Two subclones of B9-2 cells transduced with pBabeNeo-C/EBPα-ER(B9res), B9-2/Neo and Vec/Neo cells were subjected to Western blotting for C/EBPα and β-actin (left). B9-2/Neo, B9-2/αER-1, and B9-2/αER-2 cells cultured in G-CSF with estradiol were subjected to Wright-Giemsa staining on G6, the day of maximal differentiation (center). RNAs prepared in IL-3 or G-CSF with simultaneous addition of estradiol were analyzed for Mpo expression (right, n = 3).
Figure 8.
Quantitative RT-PCR analyses from RNA samples used for global gene expression analysis.
RNA samples from replicate pLKO.1 and B9 transductions were subjected to quantitative RT-PCR in triplicate for indicated mRNAs relative to β-actin RNA. Expression in pLKO.1 vector samples was set to 1.0 on average in each experiment, and mean Relative Expression values in the two B9 samples are shown.
Table 1.
Selected mRNAs decreased or increased in Lin− marrow cells by Cebpa shRNA*.
Table 2.
Top pathways with affected genes altered by Cebpa knockdown*.
Figure 9.
Myeloid CFUs and RNAs expression in Lin−Sca-1−c-Kit+ murine marrow cells expressing G-CSF versus M-CSF receptor.
A) FACS analyses demonstrating isolation of Lin−Sca-1−c-Kit+ GCSFR(GR)+MCSFR(MR)− and Lin−Sca-1−c-Kit+GCSFR(GR)−MCSFR(MR)+ populations. B) The number of CFU-M, CFU-G, or CFU-GM obtained per 1E4 cells plated in methylcellulose culture with IL-3, IL-6, and SCF is shown (n = 3). C) Total cellular RNAs were subjected to quantitative RT-PCR for indicated mRNAs (n = 3).