1. by Qian-fei Wang, and Alan D. Friedman
CCAAT/enhancer-binding proteins are required for granulopoiesis independent of their induction of the granulocyte colony-stimulating factor receptor
by Qian-fei Wang, and Alan D. Friedman
Blood
Volume 99(8):
April 15, 2002
©2002 by American Society of Hematology

2. Effect of C/EBPβ or C/EBPδ on granulopoiesis
Effect of C/EBPβ or C/EBPδ on granulopoiesis.C/EBPβ or C/EBPδ induces granulopoiesis.
Effect of C/EBPβ or C/EBPδ on granulopoiesis.C/EBPβ or C/EBPδ induces granulopoiesis. (A) Total cellular proteins corresponding to 1 × 106 cells, from subclones transduced with pBabePuro-C/EBPβWT-ER (βWT-ER), pBabePuro-ER (ER), or pBabePuro-C/EBPδWT-ER (δWT-ER) or from parental 32D cl3 cells (32D) were subjected to Western blotting with the use of a human ER antiserum. The position of a truncated C/EBPβWT-ER isoform is indicated by an asterisk. (B) 32D-C/EBPβWT-ER-1 cells, 32D-C/EBPβWT-ER-2 cells, and 32D-ER cells were exposed to 1 μM estradiol for 0, 1, 2, or 3 days. Total RNAs prepared each day were subjected to Northern blotting for MPO, LF, PU.1, and β-actin by sequential probing of the same blot. (C) 32D-C/EBPδWT-ER-1 and 32D-C/EBPδWT-ER-2 cells were analyzed similarly.
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

3. An inducible dominant inhibitor of C/EBP-regulated genes
An inducible dominant inhibitor of C/EBP-regulated genes.(A) KRAB-C/EBPα-ER (KαER) and KRAB-C/EBPα-1,2Val-ER (KVER) are diagrammed.
An inducible dominant inhibitor of C/EBP-regulated genes.(A) KRAB-C/EBPα-ER (KαER) and KRAB-C/EBPα-1,2Val-ER (KVER) are diagrammed. The KRAB segment is a transrepression domain derived from Kox-1. The C/EBPα segment contains the C/EBPα DNA-binding domain, but not its trans-activating domains. All C/EBP family members have a common DNA-binding consensus, and it is therefore expected that each will be inhibited by KαER. The ER segment is responsive to 4HT. In KVER, mutation of 2 leucines (L) to valine (V) prevents DNA binding. (B) Total cellular proteins corresponding to 1 × 106 cells, from 32D cl3 cells or from subclones transduced with pBabe-KVER or pBabe-KαER, were subjected to Western blotting with the use of a murine ER antiserum. (C) First, 5 × 106 32D–KαER-1 and 32D–KVER-2 cells proliferating in IL-3 were transfected with 20 μg p(C/EBP)2TKLUC and 1.0 μg pCMV-βGal with the use of diethylaminoethyl-dextran. One-half of each culture was then treated with 200 nM 4HT and the other half with 0.1% ethanol. Luciferase and βGal activities were assessed 2 days later. The ratio of reporter activity (activity with 4HT/activity without 4HT), normalized to the internal control, is shown for each condition (mean and SE from 2 experiments).
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

4. Effect of C/EBP when 32D cl3 cells are cultured in G-CSF
Effect of C/EBP when 32D cl3 cells are cultured in G-CSF.C/EBP inhibition leads to apoptosis without differentiation when 32D cl3 cells are cultured in G-CSF.
Effect of C/EBP when 32D cl3 cells are cultured in G-CSF.C/EBP inhibition leads to apoptosis without differentiation when 32D cl3 cells are cultured in G-CSF. (A) First, 2 × 10432D–KαER-1 (circles) or 32D–KVER-2 (squares) cells were cultured in IL-3 either without 4HT (open) or with 4HT (filled), and viable cell counts were obtained daily. Results of a typical experiment are shown (top). Then, 2 × 105 32D–KαER-1 or 32D–KVER-2 cells were transferred to G-CSF without or with 4HT, and viable cell counts were again obtained daily (bottom). (B) Morphology of 32D–KVER-2 cells in IL-3 and after transfer to G-CSF for 2, 7, or 12 days (G2, G7, G12) with 4HT. Wright-Giemsa stain, original magnification ×100. (C) Morphology of 32D–KαER-1 cells in IL-3, after transfer to G-CSF for 2, 7, or 13 days (G2, G7, G13) without 4HT, or in G-CSF for 1 or 2 days (G1, G2) with 4HT. Wright-Giemsa stain, original magnification ×100. (D) Total cellular RNAs were prepared from 32D–KαER-1 or 32D–KVER-2 cells proliferating in IL-3 or 1 day after transfer to G-CSF, without or with 4HT. These RNAs, 20 μg per lane, were subjected to Northern blot analysis for MPO and β-actin.
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

5. Effect of C/EBP inhibition on endogenous and exogenous G-CSFR expression.Inhibition of C/EBPs reduces endogenous, but not exogenous, G-CSFR expression.
Effect of C/EBP inhibition on endogenous and exogenous G-CSFR expression.Inhibition of C/EBPs reduces endogenous, but not exogenous, G-CSFR expression. (A) Total cellular RNAs were prepared from 32D–KαER-1 and 32D–KVER-2 cells proliferating in IL-3, after exposure to 4HT or the ethanol vehicle for 2 days. These RNAs were subjected to Northern blotting for G-CSFR and β-actin. (B) The 32D–KαER-1, 32D–KVER-2, 32D-KαER/GR, and 32D-KVER/GR cells were cultured with 4HT or the ethanol vehicle for 2 days. Then, 2 × 106 cells from each culture were incubated with biotin–G-CSF in the absence (thick lines) or presence (thin lines) of 1000-fold excess unlabeled G-CSF. The cells were then incubated with streptavidin-PE, fixed, and subjected to FACS analysis.
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

6. Effect of C/EBP inhibition on differentiation in response to exogenous G-CSFR.Inhibition of C/EBPs blocks and even reverses differentiation in response to exogenous G-CSFR.
Effect of C/EBP inhibition on differentiation in response to exogenous G-CSFR.Inhibition of C/EBPs blocks and even reverses differentiation in response to exogenous G-CSFR. (A) First, 2 × 10532D-KαER/GR cells were transferred to G-CSF without 4HT (○) or with 4HT (●). Viable cell counts performed daily are shown. (B) Cells from these same cultures were cytospun and subjected to Wright-Giemsa staining. Cells are shown in IL-3, after transfer to G-CSF in the absence of 4HT for 2, 4, or 6 days (G2, G4, G6), or after transfer to G-CSF in the presence of 4HT for 1, 2, 4, or 8 days (G1, G2, G4, G8). Original magnification ×100. (C) The 32D-KαER/GR cells were transferred to G-CSF without or with 4HT. A subset of cells cultured in the absence of 4HT for 3 days were then exposed to 4HT (−/+ d3). Total cellular RNAs prepared on days 0, 1, 2, 3, 4, 5, and 6 were subjected to Northern blot analysis, 20 μg per lane, for MPO, lysozyme (Lys), LF, C/EBPα, C/EBPβ, C/EBPε, and β-actin.
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

7. Effect of C/EBP inhibition on induction of CD11b or PU. 1 by G-CSF
Effect of C/EBP inhibition on induction of CD11b or PU.1 by G-CSF.Inhibition of C/EBPs does not prevent induction of CD11b or PU.1 by G-CSF.
Effect of C/EBP inhibition on induction of CD11b or PU.1 by G-CSF.Inhibition of C/EBPs does not prevent induction of CD11b or PU.1 by G-CSF. (A) KαER/GR cells were cultured in G-CSF with and without 4HT, and the expression of CD11b was assessed by FACS analysis daily. Results from a representative experiment on days 0 and 5 are shown. (B) The 32D-KαER/GR2 cells were cultured in G-CSF without or with 4HT, and total cellular RNAs prepared on days 0, 2, 4, and 7 were subjected to Northern blotting for MPO, PU.1, and β-actin.
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

8. Effect of C/EBP inhibition on the growth of myeloid colonies from murine marrow.Inhibition of C/EBPs prevents the growth of myeloid colonies from murine marrow.
Effect of C/EBP inhibition on the growth of myeloid colonies from murine marrow.Inhibition of C/EBPs prevents the growth of myeloid colonies from murine marrow. (A) FACS analysis of murine marrow isolated from 5-FU–treated mice either untransduced (left) or transduced (right) with MIG-KαER. Forward scatter is shown on the y-axis, and GFP expression is shown on the x-axis. Particles with fewer than 280 units of forward scatter were excluded from this analysis, as they are likely to represent cell debris. The gate set for isolation of GFP+ cells from the transduced marrow is also shown. Note that 19% of the transduced cells were GFPbright and that cells with modest or low levels of GFP expression were not collected. (B) The ratio, with and without 4HT, of myeloid CFUs obtained in response to the indicated cytokines, for untransduced or transduced marrow cells, and of erythroid burst-forming units (BFU-Es) from fetal liver cells is shown for a typical experiment. G indicates G-CSF; M, M-CSF; GM, GM-CSF; IL3, IL-3; and Epo, erythropoietin and insulin. An average of 13 CFU-Gs, 130 CFU-Ms, and 23 CFU-GMs per 8 × 103 GFP+-transduced cells were obtained in response to GM-CSF, in the absence of 4HT. BFU-E yields from transduced fetal liver cells averaged 96 per 4 × 104transduced cells.
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology

9. Effect of C/EBPs in cycloheximide versus actinomycin D on induction of lysozyme and C/EBPε.Lysozyme and C/EBPε are induced by C/EBPs in cycloheximide but not actinomycin D. The 32D-C/EBPαWT-ER (32D–αWT-ER) or 32D–C/EBPβWT–ER-1 (32D–βWT-ER) cells proliferati...
Effect of C/EBPs in cycloheximide versus actinomycin D on induction of lysozyme and C/EBPε.Lysozyme and C/EBPε are induced by C/EBPs in cycloheximide but not actinomycin D. The 32D-C/EBPαWT-ER (32D–αWT-ER) or 32D–C/EBPβWT–ER-1 (32D–βWT-ER) cells proliferating in IL-3 were exposed to 1 μM estradiol (Est), 50 μg/mL cycloheximide (CHX), both, or neither for 8 hours. The 32D–C/EBPαWT-ER cells were also cultured with actinomycin D (Act) with and without estradiol. Total cellular RNAs were then isolated and subjected to Northern blotting for lysozyme, C/EBPε, and MPO. An ethidium stain of ribosomal RNAs is also shown as a control for RNA integrity and loading (bottom panels).
Qian-fei Wang, and Alan D. Friedman Blood 2002;99:
©2002 by American Society of Hematology