Article Figures & Data
Figures
- Fig. 1.
Expression levels of RARs and RXRs during the granulocytic differentiation of ML-1 cells. Poly(A)+ RNA samples were prepared from cells treated with 10−7 m ATRA and 0.1 ng/ml GM-CSF for the times indicated. Northern blot analysis using 4 μg of poly(A)+ RNA was performed as described in “Materials and Methods.” Radioactivities of the bands on autoradiograms were measured using a BAS1500 image analyzer (Fuji Film, Kanagawa, Japan).
- Fig. 2.
A, expression level of RARα, RARγ, and RXRα in ML-1 cells treated with 10−7 m ATRA or 0.1 ng/ml GM-CSF for the times indicated. Northern blots were performed as described in Fig. 1 <$REFLINK> . B, Western blotting analysis of RARα and RXRα in ML-1 cells treated with GM-CSF. Cultures were treated with 0.1 ng/ml GM-CSF for the times indicated. Protein lysates were prepared, and Western blots were performed as described in “Materials and Methods.”
- Fig. 3.
Gel mobility shift assay using a labeled DR5 probe. The labeled probe was incubated with nuclear extracts from untreated ML-1 cells (Lane 1) or from ML-1 cells treated with GM-CSF for 3 (Lanes 2, 5, 6, 7, 8, and 9), 6 (Lane 3), and 24 (Lane 4) h. Competition experiments were performed by incubation with cold RAR gel shift oligonucleotide (Lane 5) or mutant oligonucleotide (Lane 6) in a 50-fold excess. For the supershift experiment, nuclear extracts were treated with RARα antibody (Lane 7), RXRα antibody (Lane 8), and rabbit serum (Lane 9). Bottom arrow (A) indicates the position of the DNA-protein complexes. Upper arrows (B and C) indicate the shifted complex formed in the presence of each antibody.
- Fig. 4.
Expression level of CD38 in ML-1 cells treated with 10−7 m ATRA and/or 0.1 ng/ml GM-CSF for the times indicated. Northern blot analyses using 4 μg of poly(A)+ RNA were performed as described in Fig. 1 <$REFLINK> . The exposure time is 6 h. All filters were exposed to the imaging plate at the same time.
- Fig. 5.
NBT-reducing activity and expression level of RARα in various cell lines treated with 10−7 m ATRA and/or 0.1 ng/ml GM-CSF. A, effect of 10−7 m ATRA and/or 0.1 ng/ml GM-CSF on the induction of NBT-reducing activity of various cell lines. NBT-reducing activity was determined as described in “Materials and Methods.” Values are the means ± SE of triplicate determinations. B, expression level of RARα in various cell lines treated with 0.1 ng/ml GM-CSF for the times indicated. Northern blots were performed as described in Fig. 1 <$REFLINK> .
- Fig. 6.
Alteration of expression level of RARα by GM-CSF in peripheral blood cells from AML patients. Total RNA samples were prepared from nontreated cells (Lanes 1, 3, and 6) and cells treated with 0.1 (Lanes 2, 4, and 7) or 1 ng/ml (Lanes 5 and 8) GM-CSF for 3 h. RT-PCR was performed as described in “Materials and Methods.” The PCR products were separated on a 1.5% agarose gel in Tris-acetate-EDTA buffer and stained with ethidium bromide.
Tables
- Table 1
Morphological changes in KG-1 and THP-1 cells after treatment with ATRA and/or GM-CSF for 9 days
Morphological maturation was classified according to the nuclear shape as blast, myelocyte, banded, and segmented. Differential counts were performed under a microscope among a minimum of 200 cells. Morphological changes were determined as described in “Materials and Methods.” Values are the mean ± SD of triplicate determinations.
Inducer Myeloid cell type (% of total cells ± SD) Blasts Myelocytes Banded neutrophils Segmented neutrophils KG-1 None 85.8 ± 2.0 14.2 ± 2.0 0.0 0.0 GM-CSF (0.1 ng/ml) 88.0 ± 3.6 12.0 ± 3.6 0.0 0.0 ATRA (10−7 m) 24.7 ± 2.4 50.0 ± 2.9 16.0± 2.7 9.3± 0.6 GM-CSF+ ATRA 7.0 ± 1.8 21.0 ± 1.8 29.7± 2.1 42.3± 2.4 THP-1 None 98.7 ± 0.2 1.3 ± 0.2 0.0 0.0 GM-CSF (0.1 ng/ml) 90.7 ± 0.8 9.3 ± 0.8 0.0 0.0 ATRA (10−7 m) 12.7 ± 2.2 31.1 ± 1.3 38.5± 2.9 17.7± 0.6 GM-CSF+ ATRA 4.7 ± 2.1 17.2 ± 3.0 28.3± 4.3 49.8± 3.2
Volume 60, Issue 16
