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Blood, Vol. 111, Issue 9, 4706-4715, May 1, 2008
IL-21 is expressed in Hodgkin lymphoma and activates STAT5: evidence that activated STAT5 is required for Hodgkin lymphomagenesis
Blood Scheeren et al.
111: 4706
Supplemental materials for: Scheeren et al
Files in this Data Supplement:
- Figure S1. Immunoblot analysis for IL-21 and actin as a loading control was performed on CD4+ T cells activated with CD3/CD28 beads as a postive control for the IL-21 antibody (JPG, 8.84 KB)
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- Figure S2. Cytospins of CA-STAT5b+ peripheral blood B cells were prepared and a Giemsa staining was performed to visualize the nuclei of the cells (JPG, 4.01 KB)
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- Figure S3. Protein levels of BCL6 in the nuclear fraction in two HL cell lines (L1236 and L591) and two CA-STAT5b+ tonsil cell line that grew independent of CD40 activation (JGP, 8.76 KB)
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- Figure S4. The HL cell line L591 was transduced with IκBSR or a control virus and subsequent sorted (JPG, 5.28 KB)
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Western blot analysis was performed for I B on the transduced cells.
- Figure S5. L428 was retrovirally transduced with a vector expressing a siRNA targeting STAT5 or Renilla as a control (JPG, 4.86 KB)
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These retroviral constructs contained a GFP expression cassette. Immunoblots for STAT5 and actin were performed on GFP purified cells, showing efficient STAT5 protein reduction.
- Figure S6. Genescan and RT-PCR analysis of clonality in PB B cells expressing CA-STAT5b-ER and CA-IKK2 (JPG, 30.4 KB)
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CD19+ B cells were transduced with CA-STAT5bER-DNGFR and grown with CD40L-L cells, 4-HT, and IL-2+IL-4 for 49 days. These cells were then transduced with CA-IKK-GFP, removed from CD40L-L cells, but maintained with 4-HT and cytokines for 48 days, for a total of 97 days in culture. (A) Genescan analysis of genomic DNA at Day 97. (C) RT-PCR analysis of Vh and Cµ usage at Day 97. (B, D). CD19+ B cells were transduced with CA-STAT5bER-DNGFR and CA-IKK-GFP and cultured without CD40L-L cells, but with 4-HT and cytokines for 41 days. (B) Genescan analysis of genomic DNA at Day 41. (D) RT-PCR analysis of Vh and Cµ usage at Day 41.
- Figure S7. Ramos cells were electroporated with control luciferase vector (pBasic) or a retinoic acid-responsive elements luciferase vector (RARE-luciferase) with responsive element in combination with a pSuper vector that knocks down STAT5 using siRNA or an empty control (n = 2) (JPG, 17.6 KB)
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At 72 h firefly luciferase activity was measured, normalized to the co-transfected Renilla luciferase activity.
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