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Blood, Vol. 112, Issue 7, 2750-2760, October 1, 2008
Igbp1 is part of a positive feedback loop in stem cell factor–dependent, selective mRNA translation initiation inhibiting erythroid differentiation
Blood Grech et al.
112: 2750
Supplemental materials for: Grech et al
Files in this Data Supplement:
- Document 1. Supplemental materials and methods (PDF, 71.9 KB)
- Table S1. Description of samples used for array hybridizations (PDF, 1.62 MB)
- Table S2. Functional characteristics of translationally controlled genes selected for functional studies (PDF, 1.27 MB)
- Table S3. Primers used for Q PCR (PDF, 555 KB)
- Table S4. Array results (XLS, 16 MB)
- Table S5. Fold change expression in I/11 and R10 individual samples, plus weighted averages, following factor deprivation and restimulation with Epo/SCF, and during differentiation (XLS, 233 KB)
- Figure S1. Different patterns of growth factor dependent mRNA recruitment to polysomes as revealed by cluster analysis (JPG, 82.8 KB)
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The mean hybridisation intensity of total and polysome bound (pb) mRNA derived from factor deprived (NF) and Epo/SCF restimulated cells (ES) was plotted for several probe sets representing genes in each cluster of figure 1. The bars represent the normalized geometric mean of the hybridisation intensity of 4 replicates and the error bars represent the standard error as calculated by Rosetta Resolver software. Heat Map cluster 1 represented by Nap1l1 and Cnih (A,B), cluster 3 by Actg1 (C); cluster 2 and 4 by Pscd3, Ndst2 and Txnip (D-F); and cluster 5 by Hnrpa1 and Uhmk1 (G, H).
- Figure S2. Signalling dependent expression of Igbp1 and Uhmk1 protein (JPG, 91 KB)
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(A) Inhibitors of translation (cycloheximide 50g/ml; ChX) and transcription (Actinomycin D 10g/ml; ActinoD) were added for 2, 4, 6 and 8 h to proliferating I/11 cells and cell ysates were probed for expression of Igbp1, Uhmk1 and Fli1 using specific antibodies. Intensities were measured and plotted as a foldchange expression compared to no addition. The insert shows the linearity of Igbp1 detection in subsequent 2-fold dilutions of total cell lysate (r2=0.9939). Bars for ChX (black) and ActinoD (white) indicate fold change (fc) expression compared to ActinoD 2hr expression. Compared to Fli-I, expression of Igbp1 and Uhmk1 is more sensitive to translation inhibition. (B) I/11 cells were factor deprived for 4 h and restimulated with Epo plus SCF for 2 h (stimulation) or left untreated (starvation). Expanding I/11 cells in the presence of Epo, SCF and dexamethasone are denoted as ss (steady state). Inhibitors of mTOR (rapamycin 40ng/ml; rapa) and translation elongation (ChX) were added during factor deprivation and subsequent restimulation. Bars indicate fold change of protein expression compared to expression after factor deprivation.
- Figure S3. Subcellular protein localisation of selected genes used for functional studies (JPG, 87 KB)
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Phoenix cells grown on microscope slides were fixed and stained with anti-myc tag antibodies and a FITC-labeled secondary antibody, 48h following transduction with retroviral expression vectors harbouring myc-tagged cDNA constructs of Uhmk1, Cnih, Igbp1, mEd2, Hnrpa1, Rnf138 or Nap1l1. Imaging of the cells was done with a confocal scanning microscope (Zeiss). A 10m bar in the Rnf138 panel indicates the scale.
- Figure S4. Expression of ectopic Igbp1 in single cell derived clones (JPG, 75.4 KB)
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I/11 cells were transduced with the coding sequence of Immunoglobulin binding protein 1 (Igbp1), tagged with the myc epitope. The single cell derived clones were expanded in medium supplemented with 0.5U/ml Epo, 100ng/ml SCF and 10−6M dexametasone. Western blots with total lysates were stained with antibodies specifically binding the myc epitope. The first 2 lanes are empty vector clones, followed by lysates of clones #2, #5, #9, used in figure 4 and clones #12 and #17 used in Fig. 3.
- Figure S5. Igbp1 expression is PI3K dependent and eIF4E level sensitive (JPG, 81.2 KB)
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I/11 cells were factor deprived for 4 h and restimulated with Epo (5U/ml) plus SCF (100ng/ml) for 2 h (ES) or left untreated (NF). In addition, I/11 cells were factor deprived and restimulated with Epo plus SCF for 2 h in presence of 10µM LY294002 (ESly). The same conditions were used for single cell derived I/11 clones overexpressing the translation initiation factor eIF4E. Western blots with total lysates were stained with antibodies specifically binding Igbp1 and actin (loading control).
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