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Blood, Vol. 106, Issue 13, 4114-4123, December 15, 2005
The E3 ubiquitin-protein ligase Triad1 inhibits clonogenic growth of primary myeloid progenitor cells
Blood Marteijn et al.
106: 4114
Supplemental materials for: Marteijn et al
Files in this Data Supplement:
- Figure S1. Triad1 transcript, protein and protein conservation (PDF, 84.7 KB) -
A. Human Triad1 cDNA and protein sequence. The translational start site (cDNA position 145) is accompanied by a Kozak consensus sequence (underlined) and followed by a 1479 bp open reading frame. A poly-adenylation signal is present at cDNA position 3869 (bold). Typical Triad1 protein domains are a 55 aa N-terminal acidic (aa pos 4-58, underlined), two RING finger (aa positions 139-188 and 297-326, bold, boxed with cys/his residues essential to the RING finger structure encircled), a cysteine-rich (DRIL, aa positions 228-270, bold, boxed and cys/his residues encircled) and two C-terminal coiled coil (aa pos 351-399 and 442-487, shaded) domains. The two putative NLS (aa pos 242-248 and 438-444) are in bold and underlined. Alternative splicing occurs at cDNA position 399 (vertical arrow, see also Figure 1b, lane 2). B. Triad1 amino acid conservation. Indicated are the acidic(*), ring finger ($), DRIL (#) and coiled coil (^) regions. Genbank accession numbers of murine, D. melanogaster (CG-5709A) and C. elegans (4G323) proteins are respectively, XP_484030, NP_477374, and NP_500829. The putative human/mouse NLS PRARRVQ and PRKKLFE are respectively present at aa positions 242/241 and 438/437, whereas the C. elegans NLS PEPKRKL and KPKR are respectively present at aa positions 111 and 233. C. Extremely high C-terminal conservation (identity between human Triad1 and other species is indicated in percentages). Note that despite the overall low N-terminal conservation, all cysteine and histidine residues characteristic for the N-terminal RING finger structure and DRIL domain are conserved (see also supplemental Figure 1b). A = acidic, R = RING finger, D = DRIL domain, R2 = RING2 finger, CC = coiled coil.
- Figure S2. Triad1 expression in hematopoietic subsets measured with flowcytometer (PDF, 118 KB) -
A. Representative CD45/side scatter plot with CD34+ cells in pink. Triad1 expression within CD45+CD34+ compartment is indicated. B. Identical sample as in A with CD3+ cells in pink. Triad1 expression within CD45+CD3+ compartment is indicated. C. Identical sample as in A with CD14+ cells indicated in pink. CD14+ compartment is divided into two subcompartments Z and AD representing respectively, fractions with more pro-monocytes and mature monocytes based on CD45 and side scatter plot (Van Lochem et al, Cytometry 60B:1-13, 2004). Triad1 expression in both compartments is indicated and the mean Triad1 expression in these two fractions (Mo1 and Mo2) is indicated in Figure 3c. D. Identical sample as in with CD15+ cells indicated in pink. The CD15+ compartment is divided into three subcompartments AB, AD and AE representing respectively immature myelocytic cells (including promyelocytes), more committed myelocytic cells (including myelocytes/metamyelocytes) and mature granulocytes (neutrophils) (Van Lochem et al, Cytometry 60B:1-13, 2004). Triad1 expression in these compartments is indicated and the mean Triad1 fluorescence in these three fractions (Gr1–3) is indicated in Figure 3c.
- Figure S3 (PDF, 99 KB) -
A. Representative example of GFP expression in pLZRS-Triad1 transduced primary murine bone marrow cells. Sorted fractions (Triad1 I, II and III) with increasing GFP positivity are indicated. B. Representative result of CFU-GM colony size derived from Triad1 and empty vector (EV) transduced cells. Treatment of Triad1 transduced cells with 10-8 M MG132 prior to cell seeding results in significantly larger colonies. C. Representative DNA histograms of empty vector (EV) and Triad1 transduced cells after 3 days of culture are indicated (see Figure 7b).
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