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Blood, Vol. 112, Issue 5, 1981-1992, September 1, 2008
Id1 is a common downstream target of oncogenic tyrosine kinases in leukemic cells
Blood Tam et al.
112: 1981
Supplemental materials for: Tam et al
Files in this Data Supplement:
- Table S1. The expression data of the microarray used in this study (XLS, 29.6 MB)
- Table S2. The descriptions of the names used in Table S1 (XLS, 14 KB)
- Figure S1. ID1 mRNA expression in adult AML patients with normal karyotype according to FLT3-ITD status (JPG, 39.6 KB)
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Among 116 AML patients with normal cytogenetics (Valk et al. N Eng J Med 2004), ID1 expression levels were significantly higher in cases with FLT3-ITD mutations (P < .0001 as assessed using the unpaired t test). In these diagrams, the upper edge of the box indicates the 75th percentile of the data set; the lower edge indicates the 25th percentile; the line in the box indicates the median value of the data. The vertical lines indicate the upper and lower 25% of the data, respectively; the ends of the vertical lines indicate the minimum and maximum data values.
- Figure S2. ID1 mRNA expression in adult patients with acute promyelocytic leukemia according to FLT3-ITD status (JPG, 39.6 KB)
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Among 33 patients with acute promyelocytic leukemia (L. Bullinger, unpublished results), there was a trend towards higher ID1 expression levels in cases with FLT3-ITD mutations (P = .077 as assessed using the unpaired t test). In these diagrams, the upper edge of the box indicates the 75th percentile of the data set; the lower edge indicates the 25th percentile; the line in the box indicates the median value of the data. The vertical lines indicate the upper and lower 25% of the data, respectively; the ends of the vertical lines indicate the minimum and maximum data values.
- Figure S3. Flow cytometric analysis of GFP positive cells (JPG, 61.2 KB)
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K562 cells or Molm-14 cells transduced with lentiviral vector expressing either GFP alone, GFP with siRNA-GL, or GFP with siRNA-Id1. The transduction efficiency is over 95% of the cell population.
- Figure S4. K562 cells (JPG, 47.2 KB)
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(A) Western blot analysis of Id1 and p27 expression in K562 cells. Protein lysates were prepared from stable clones transfected with sense Id1 (K562pBabe-Id1), empty vector (K562pBabe), or antisense Id1 (K562pBabe-Id1AS). S1, S2, P1, P2, K1, and K2 denote the two individual clones of each transfection. To confirm equal loading, the membrane was stripped and re-blotted with RACK1 antibody. (B) Growth curve of K562 cells measured by CellTiter 96 Aqueous One Solution Cell Proliferation Assay. Note that the two tranfectants of pBabe-Id1AS grow much slower than the K562 cells transfected with pBabe or pBabe-Id1.
- Figure S5. Western blot analysis of Id1 expression in TonB/ Fl3-ITD upon treatment of PI3 kinase inhibitor, Jak inhibitor and MAPK kinase inhibitor (JPG, 73.3 KB)
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TonB/Flt3-ITD cells were grown in RPMI 1640 medium supplemented with IL3 (2ng/ml). Prior to treatment with inhibitors, cells were washed and resuspended in medium either with IL3 (Fig. S5, Right Panel) or without IL3 (Fig. S5, Left Panel) for 6 hours. Six hours later, 2µg/ml of doxcycline was added to cells maintained in medium without IL3. Cells were then maintained either untreated or treated with 10 µM of LY 294002 (Fig. S5A), 1µM of JAK inhibitor (Fig. S5B), 10µM of PD 98059 (Fig. S5C) for 36 hours. All of the inhibitors are purchased from Calbiochem (Gibbstown, NJ) and used according to the manufacturer’s instructions. Western blot was probed with anti-ID1 antibody (Santa Cruz Biotechnology, Santa Cruz, CA), anti-phosphorylated Stat5 antibody, anti-phosphorylated S6 kinase antibody, or anti-phosphorylated MAPK antibody as indicated. The blot was re-stripped and probed with Stat antibody, S6 kinase antibody, and MAPK antibody. The antibodies were purchased from Cell Signaling, Danvers, MA.
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