Legend to supplementary figures:
Supplementary Fig. 1. In vitro biochemical properties of the TEL-ARNT fusion protein.
TEL-ARNT interacts with HIF1
Radiolabelled proteins were immunoprecipitated using the indicated antibodies. Lanes 1 to 8
are control experiments. Lanes 1, 4, 7, 9, 11 correspond to the reaction before
immunoprecipitation. Antibody against ARNT precipitates HIF1 only when ARNT is coexpressed (lane 10). In the same way, antibodies against either TEL or ARNT precipitate
HIF1 only when TEL-ARNT is co-expressed (lanes 12 and 13).
Each construct was in vitro translated in the presence of [35S] methionine in a TNT-coupled
transcription/translation system (Promega). Immunoprecipitation assays were performed with
two rabbit antisera raised against the amino-terminal part of TEL, and against the carboxyterminal part of ARNT (amino acid residues 499-789 of the human ARNT protein: serum 76).
In these experiments, truncated ARNT and TEL-ARNT protein were used to facilitate size
discrimination. Ten ul of each in vitro translation was incubated with 2 ul of antiserum, in
500ul of immunoprecipitation buffer (25mM Tris pH7.4, 20% glycerol, 2mM EDTA, 5mM
DTT, 0.2% NP40, 100mM KCL), with protease and phosphatase inhibitors and 100 g of
BSA and 12 picomoles of HRE probe for dimer stabilization. After gentle mixing for 3 hours
at 4°C, the immunocomplexes were isolated using protein A Sepharose. Samples were diluted
in Laemmli buffer and loaded on denaturating acrylamide gels.
Supplementary Fig. 2. Transcriptional activity of ARNT and TEL-ARNT on the human
VEGF promoter
Comparison of transcriptional regulation of vEGF promoter by ARNT and TEL-ARNT in
response to low oxygen tension and to DFO. In low oxygen tension ARNT expression
enhances the response of the human VEGF promoter, with respect to an empty vector
(pcDNA) transfection ; whereas TEL ARNT does not enhance transcription by on the
contrary exert a small negative effect. A qualitatively similar response is observed in the
presence of DFO, which mimics hypoxia. No effect is seen in normixic conditions. RLU :
relative light units. Error bars represent standard deviation.
Supplementary Fig. 3. Low oxygen tension stimulates proliferation and differentiation
of human CD34 cells along erythrocytic and megakaryocytic pathways. Hi: High
oxygen tension, Lo: Low oxygen tension
We stimulated CD34+ cells along the erythroid lineage. For that purpose, CD34+ cells
were grown for 4 days in medium supplemented with IL3, IL6 and SCF and then either
maintained in high oxygen tension or shifted to low oxygen tension conditions. Epo
was added three days after shift and cells were incubated for an additional three days.
The results of Sup Figure 3a show that low oxygen tension resulted in a higher
proliferation rate than in high oxygen tension conditions. Cell differentiation was
investigated by assessing the expression of GPA and CD36, two erythroid specific
antigens. Incubation in 1% O2 resulted in a marked enhancement of cell differentiation
with respect to 20% O2 conditions as judged by a three fold enhancement of GPA
expression at day 5 (Sup Figure 3b) and CD36 expression (data not shown).
To monitor differentiation along the megakaryocytic pathway, CD34+ cells were
differentiated as described in Materials and methods. Cell surface expression of CD42b
(Sup Figure 3d) and CD41a (data not shown) was examined. Ploïdy of single cells was
also determined by morphological analyses (Supplementary Figure 3d). All data
showed a clear increase in both cell numbers (three time in absolute cell numbers at
day 6; Supplementary Figure 3c) and differentiation as shown by a 4 fold increase in
the percentage of cells expressing CD42 at day 6 (Figure 3d) in low oxygen tension
with respect to high oxygen tension conditions. Finally, the number of cells containing
two or three nuclei was also two times higher in low versus high oxygen tension
conditions at day 6 (Supplementary Figure 3e).
(a) Total cell counts during differentiation along the erythrocytic pathway.
(b) FACS analyses during differentiation along the erythrocytic pathway show an
increase in number of GPA positive cells. Cells were analyzed by FACS after staining
with anti-GPA monoclonal antibody from the day 0 (switch in low oxygen tension
conditions) and at the indicated days.
(c) Total cell counts during differentiation along the megakaryocytic pathway.
(d) FACS analyses of megakaryocytic differentiation show an increase in number of
CD42b positive cells. Cells were analyzed by FACS after staining with antiCD42b
monoclonal antibody
(e) Morphological analyses of megakaryocytic differentiation. Cytospins from cells were
scored for megakaryocytic differentiation on the basis of number of nuclear lobules.
For erythroid differentiation, sorted CD34+ cells were cultured in serum-free RTM
medium (Mabio-international Tourcoing France) complemented with interleukin (IL)-3
(10 ng/ml), IL-6 (10 ng/ml), Stem Cell Factor (SCF) (25 ng/ml) for 4 days. At this time
(day 0) cells were seeded either in high (20%) or low (1%) oxygen tension conditions
in the same medium. Erythropoietin (Epo) (2 unit/ml) was added at day 3. Cell
numbers were determined in duplicate, in two separate wheels. Cell differentiation was
monitored by FACS analyze (FACS calibur BD) using Phycoerythrin (PE)-coupled
antibody to Glycophorin A (GPA) and Fluorescein isothiocyanate (FITC)-coupled
antibody to CD36.
For megacaryocyte differentiation, sorted cells were cultured in IMDM complemented
with 15% BIT9500 medium (Stem Cell Technologies) and Thrombopoietin mimetic
peptide (TPOmp 20nM) (Genosys Biotechnologies, St Quentin en Yvelynes, France),
TPO (450U/ml) (Zymo Genetics), SCF (5ng/ml) for 3 days. At this time (day 0) cells
were seeded either in high or low oxygen conditions in the same medium. Cell
concentration was determined as for erythroid differentiation. Differentiation was
monitored using FITC-antiCD41a and PE-antiCD42b cell surface marker expressions
and by morphological analyses.
Supplementary Fig. 4. Low oxygen tension stimulates proliferation of human CD34 cells
(A) Total cell counts during differentiation along the granulocytic pathway.