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Blood, Vol. 112, Issue 4, 1056-1067, August 15, 2008
Differential use of SCL/TAL-1 DNA-binding domain in developmental hematopoiesis
Blood Kassouf et al.
112: 1056
Supplemental materials for: Kassouf et al
Files in this Data Supplement:
- Document 1. Supplemental results, materials, and methods (PDF, 122 KB)
- Figure S1. Characterisation of the SCLRER protein (JPG, 86.1 KB)
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(A) Gel shift assays. Nuclear extracts prepared from sclWt/Wt and sclRER/RER fetal livers and mouse erythroleulemia (MEL) control cells were incubated with a labelled E-box containing oligonucleotide in absence (−) or presence of anti-E12 (αE12) or anti-SCL (αSCL) antibodies, wild-type (WT oligo) or mutated (Mut oligo) cold competitor oligonucleotide. SCL/E12: retarded heterodimer. The arrow shows the supershift generated upon incubation with αSCL antibodies, the star an uncharacterised fetal liver-specific complex not present in MEL cell extracts. (B) Western Blot analysis from nuclear extracts prepared as in Figure S1A shows levels of expression of SCL and a loading control (RNA Polymerase II, Pol II). Triangles indicate serial dilutions of the input. Ratios of SCL to Pol II signal intensities are shown. (C) Immunofluorescence staining using SCL antibodies and nuclear DAPI staining on fetal liver cells derived from wild-type and homozygous sclRER/RER mutant embryos. (D) Autoradiograms show the genomic analysis of targeted homozygous (RERneo/RERneo or RERΔneo/RERΔneo) ES clones. The restriction enzymes and probes used are indicated under each blot. For schematic representation of the loci, see Figure 1B.
- Figure S2. Adult and fetal hematopoiesis (JPG, 144 KB)
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(A) Day E12.5 fetal liver cells harvested from sclWt/Wt, sclWt/RER and sclRER/RER embryos were plated in methylcellulose for colony assays. CFU-E, BFU-E, megakaryocytic (meg), mixed and myeloid colonies were scored. Error bars indicate +/− 1 standard deviation from at least 3 independent experiments. , p<0.01. (B) Purification of CMP and MEP populations. The FACS analyses show the strategy followed to isolate CMPs and MEPs from lineage-depleted bone marrow cells. Percentages of cells in the c-kithigh, CMP and MEP bone marrow cellular populations isolated from wild-type, sclWt/RER and sclRER/RER mice. Error bars indicate +/− 1 standard deviation from at least 3 independent experiments. (C) Bone marrow cells isolated from wild-type, heterozygous and homozygous two months old mice were plated in methylcellulose for colony assays, using conditions described in Materials and Methods. CFU-E, BFU-E, megakaryocytic (meg) and myeloid colonies were scored. Error bars indicate +/− 1 standard deviation from at least 3 independent experiments. *, p<0.01. (D) Recovery from induced anemia. Phenylhydrazine (PHZ)-induced stress erythropoiesis. Mice were injected with PHZ on three consecutive days. Reticulocyte count and hematocrit were assessed at regular intervals over a period of up to 26 days. Recovery of normal blood parameters was similar in wild-type, heterozygous and sclRER/RER mice.
- Figure S3. Fetal liver culture system (JPG, 140 KB)
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(A) Experimental system. (B) Morphological analyses of the cellular populations derived from wild-type, sclWt/RER and sclRER/RER E12.5 fetal liver cultures at day 0 (progenitors, Ter119−) and after 1 to 3 days of erythroid differentiation (40x objective, 10µm scale bar). Red arrows, late normoblasts are present in all the day 2 populations. In the inset, note the irregular shape of the sclRER/RER cells. (C and D) Characterisation of day 0 and day 2 fetal liver populations. (C) The proliferative capacity of the fetal liver cells was assessed over a period of 6 days in expansion (left) or 3 days in differentiation (right) cultures. The cumulative cell numbers are shown. (D) Day 0 (left) and day 2 (right) cellular populations were characterised according to cell surface marker expression by FACS analysis. The cKit+/CD71+ and cKit−/CD71+populations represent early erythroid cells; CD71+Ter119+ and CD71−Ter119+, more mature to late erythroid cells. Gr-1 and Mac-1 staining show no contamination by myeloid cells. Error bars indicate +/− 1 standard deviation from at least 3 independent experiments.
- Figure S4. Transcription factor binding and chromatin structure at the pb4.2, eklf and gpa promoters in Ter119+ cells (JPG, 148 KB)
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(A) SCL protein level in day 0 and day 2 cell populations derived from sclWt/Wt and sclRER/RER fetal liver cultures. FACS analyses of SCL-positive events are reported as percentages of SCL-expressing cells in day 0 and day 2 populations. The mean fluorescence reflects SCL expression levels in the same cell populations. Error bars show standard deviation from more than three independent experiments. *, p<0.01. (B) See legend of Figure 6 in main text.
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