1. D3: A Gene Induced During Myeloid Cell Differentiation of Linlo c-Kit+ Sca-1+ Progenitor Cells
by Sarah R. Weiler, John M. Gooya, Mariaestela Ortiz, Schickwann Tsai, Steven J. Collins, and Jonathan R. Keller
Blood
Volume 93(2):
January 15, 1999
©1999 by American Society of Hematology

2. (A) Northern blot analysis of RNA extracted from the EML and MPRO cell lines.
(A) Northern blot analysis of RNA extracted from the EML and MPRO cell lines. The blots were probed with partial cDNA clones isolated by DDRT-PCR and reprobed with human β-actin. (B) Northern blot analysis of RNA extracted from MPRO, EPRO, and EML cell lines and EML cells treated with IFNγ for 16 hours or IL-3/SCF/atRA for 6 days. The blot was probed with the 3′ UTR of the D3 gene and reprobed with G3PDH. (C) Northern blot analysis of RNA extracted from EML cells treated for 1 or 2 days with (+) or without (−) CM, SCF, IL-3, and atRA. The blot was probed with the 3′ UTR of D3 gene and reprobed with G3PDH. (D) Northern blot analysis of RNA extracted from EML cells cultured for 0 to 144 hours in IL-3/BHK CM/atRA. The blot was probed with D3 (top), MPO (center), and G3PDH (bottom).
Sarah R. Weiler et al. Blood 1999;93:
©1999 by American Society of Hematology

3. (A) Northern blot analysis of poly A+ RNA extracted from normal mouse tissues.
(A) Northern blot analysis of poly A+ RNA extracted from normal mouse tissues. The blots were probed with a D3 specific probe (top) and reprobed with human β-actin (bottom). (B) Northern blot analysis of RNA extracted from BMC, lineage-depleted BMC (Lin−), and Lin− cells purified by flow cytometry for expression of c-Kit (Lin−c-Kit+). The blots were probed with the D3 specific probe (top) and equal loading of samples was verified by ethidium bromide staining of rRNA (bottom). (C) RT-PCR analysis of D3 expression (left) in Lin− c-Kit+ Sca-1+ BMC cultured 0, 3, and 7 days in IL-3/SCF. Amplification of actin from each cDNA sample is shown (right).
Sarah R. Weiler et al. Blood 1999;93:
©1999 by American Society of Hematology

4. RT-PCR analysis of D3 expression in cell lines and in normal hematopoietic cells.
RT-PCR analysis of D3 expression in cell lines and in normal hematopoietic cells. (A) Plasmids containing the full-length cDNAs for D3 and 204 were purified and amplified using D3/204 primers, which amplifies a 505-bp band visualized by ethidium bromide staining. The amplification products were then purified and treated with (+) or without (−) Cla I. RNA extracted from EML, EML cultured in IL-3/SCF/atRA for 6 days, MPRO, and EPRO cell lines were reverse transcribed with oligo (dT), amplified with D3/204 primers, and treated with (+) or without (−) Cla I. (B) RNA from normal hematopoietic cells was purified and analyzed for D3 expression by RT-PCR as indicated above.
Sarah R. Weiler et al. Blood 1999;93:
©1999 by American Society of Hematology

5. Induction of D3 expression in differentiating bone marrow macrophages.
Induction of D3 expression in differentiating bone marrow macrophages. RNA was extracted from normal BMC treated 0 to 5 days in M-CSF in vitro as described in Materials and Methods. RNA was analyzed on Northern blots, which were probed with D3 (top), and equal loading of RNA samples was verified by ethidium bromide staining of rRNA (bottom).
Sarah R. Weiler et al. Blood 1999;93:
©1999 by American Society of Hematology

6. Induction of HGF-responsive CFU-c progenitors from EML cells.
Induction of HGF-responsive CFU-c progenitors from EML cells. EML cells were induced to undergo myeloid differentiation according to the procedures described in Materials and Methods. (A) The total number of GM-CSF–responsive colonies generated from 1 × 105 EML cells cultured in SCF/atRA with or without IL-3. (B) The total number of EPO-responsive colonies generated from 1 × 105 EML cells cultured in SCF/atRA with or without IL-3. (C) The total number of SCF-responsive colonies generated from 1 × 105 EML cells cultured in SCF/atRA with or without IL-3. (▪) SCF + RA + IL-3; (•) SCF + RA.
Sarah R. Weiler et al. Blood 1999;93:
©1999 by American Society of Hematology