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Blood, Vol. 110, Issue 6, 2027-2033, September 15, 2007
Novel RUNX1 isoforms determine the fate of acute myeloid leukemia cells by controlling CD56 expression
Blood Gattenloehner et al.
110: 2027
Supplemental materials for: Gattenloehner et al, Vol 110, Issue 6, 2027-2033
Files in this Data Supplement:
- Figure S6. Autoradiography of incorportated 32P -dCTP after reverse cDNA transcription (JPG, 85.2 KB)
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Out of a 50µl RT reaction, 2 µl radioactive cDNA were spotted on a nitrocellulose membrane, and radioactivity was measured by phopsphoimager. According to the absolute counts per minutes (cpm) of each probe, cDNA amounts were equalized for subsequent RT-PCR (last column).
- Figure S7. RT-PCR detection of RUNX1 p30 (arrow) and p24 (arrowhead) with primers specific for RUNX1/Exon 2a (UP) and RUNX1/Exon 5.4 (LP) (JPG, 28.3 KB)
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M indicates DNA size marker; 1, muscle; 2, salivary gland; 3, thymus; 4, duodenum; 5, spleen; 6, heart; 7, liver; 8, lymph node; 9, stomach; 10, brain; 11, lung; 12, kidney; 13, tonsil; 14, placenta, 15, testis; 16, skin; 17, mammary gland; 18, adrenal gland; 19, pancreas; 20, periphereal blood leucoytes; 21, colon.
- Figure S8. (JPG, 39.4 KB)
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Radiography of dried agarose gel showing representative RT-PCRs of ex vivo samples of CD56(+) (lanes 2-4, black bar) and CD56(-, red bar) (lanes 5-7) AMLs using 32P dCTP and primers specific for RUNX1 exon 2b and exon 6 (amplifying exon 2b to exon 6) and exon 2b and exon 5.4, respectively (amplifying exon 2b to exon 5.4.) There is complementary mRNA expression of RUNX1 p48 and RUNX1 p38a/p30 in CD56(+) and CD56(-) AMLs. M is the DNA size marker (in lane 1).
- Figure S9. (JPG, 54.5 KB)
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Transfection of the human AML cell lines K562, HL60, and U937 with the RUNX1 isoforms p48, p38a, p30, and p24 and subsequent semiquantitative radioactive mRNA detection of the RUNX1 target genes CD56, granzyme B, p21, IL-6, and GM-CSF by RT-PCR using gene-specific primers and 32P dCTP (radiography of dried agarose gel). In accordance with the luciferase reporter gene assays, RUNX1 p48 showed induction of mRNA expression of all RUNX1 target genes investigated, whereas RUNX1 p30 showed stable and RUNX1 p24 and p38a showed decreased expression of CD56 as compared to controls. VC indicates vector control; -, respective non-transfected cell line; M, radioactive labelled DNA size marker (32P γATP). Primers are given (see “Sequences of oligonucleotid primers” in Document 1).
- Figure S10. In vitro imaging of living K562 cells after transfection with GFP control plasmid showing transfection efficiency of 85% (JPG, 62.4 KB)
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(Left) GFP fluorescence microscopy. (Right) Brightfield light microscopy.
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