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Blood, Vol. 111, Issue 3, 1634-1643, February 1, 2008
Gene transactivation without direct DNA binding defines a novel gain-of-function for PML-RAR
Blood van Wageningen et al.
111: 1634
Supplemental materials for: van Wageningen et al
Files in this Data Supplement:
- Figure S1. Transactivation data before correction (JPG, 49.5 KB)
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Raw luciferase activaties are shown for one transactivation experiment from figure 3C. The ID1 promoter, wt or with a mutated GCbox and/or CCAATbox, was cloned in front of the firefly gene. A renilla expression construct was co-transfected for correction. Note that the correction does not qualitivily change the results.
- Figure S2. NF-Y and Sp1 are expressed in APL cells (JPG, 29.1 KB)
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MRNA expression levels of NF-Y (A) and Sp1 (B) were determined in mononuclear cells from 3 APL patients. Bone marrow samples were obtained from t(15;17) APL patients following informed consent. Mononuclear cells were isolated by density gradient centrifugation using ficoll (1.077 g/ml, Sigma). Cells were taken up in RNA-bee (ISO-TEX Diagnostics, Friendswood, USA). RNA was used as template in a RT-DNA reaction as described before (de vries br j cancer 1999). Quantitative PCR was performed with the ABI/PRISM 7700 Sequence Detection system (ABI/PE, Foster City, Ca, USA). As a reference gene we used PBGD (de vries br j cancer 1999). The primer/probe sets for detecting NF-YA and Sp1 were from Applied biosystems (Foster City, Ca, USA). PCRs were performed in universal master mix (Roche) Conditions were as follows: 10 min. 95°C followed by 45 cycles of 15 sec. 95°C and 1 min. 62°C.
- Figure S3. Transactivation of the ID1 promoter in myeloid cells (JPG, 11.5 KB)
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HL60 cells were electroporated with the 963bp ID1 promoter-luciferase reporter construct (ID1-luc) together with a control vector expressing Renilla-luciferase. In addition an empty vector or a vector coding for PML-RAR  R was transfected. Transactivation is expressed as arbitrary units and is corrected for transfection efficiency measured by Renilla-luciferase. Background luminescence of the cells transfected with only the reporter construct without nuclear receptors and in the absence of ATRA was set at 1. Cells were cultured for 8 hrs without (white bars) and with (black bars) ATRA.
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