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Blood, Vol. 112, Issue 5, 1804-1812, September 1, 2008
Spi-B inhibits human plasma cell differentiation by repressing BLIMP1 and XBP-1 expression
Blood Schmidlin et al.
112: 1804
Supplemental materials for: Schmidlin et al
Files in this Data Supplement:
- Table S1. Primers for icycler PCR (PDF, 12 KB)
- Table S2. Primers for ChIP (PDF, 14.7 KB)
- Figure S1. Detection of ectopic Spi-B/full length and Spi-B/ΔTAD expression in PB B cells (JPG, 48.3 KB)
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PB B cells transduced with LZRS-Spi-B/full-length, LZRS-Spi-B/ΔTAD or control virus were sorted for GFP expression three days after transduction and Spi-B expression was assessed by immunoblotting relative to actin levels. Asterisk indicates unspecific band. One representative experiment of two is shown.
- Figure S2. Detection of Spi-B/full length~ER and Spi-B/ΔEts~ER fusion protein expression in PB B cells (JPG, 41.1 KB)
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PB B cells transduced with LZRS-Spi-B/full-length~ER, LZRS-Spi-B/ΔEts~ER or control virus were sorted for GFP expression three days after transduction and Spi-B expression was assessed by immunoblotting relative to actin levels. One representative experiment of two is shown.
- Figure S3. Decreased levels of spliced XBP-1 upon Spi-B overexpression (JPG, 39.9 KB)
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CD19+ B cells were retrovirally transduced with constructs expressing Spi-B, BCL-6 or control-GFP. Five days after transduction and culturing in conditions promoting plasma cell differentiation (as in Figure 1), GFP+ cells were sorted. XBP-1 gene expression levels were analyzed by “classical” RT-PCR with primers spanning over the splicing region {Davies, 2003 434 /id}. One representative experiment of two is shown.
- Figure S4. The human BLIMP1 promoter is regulated by Spi-B (JPG, 44.6 KB)
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The BLIMP1 promoter-luciferase construct containing 2kb of the human PRDM1 locus upstream of the transcription start was kindly provided by A. Dent {Vasanwala, 2002 21 /id}. NIH3T3 cells were transfected with 0.1ng pCMV Renilla, 0.9µg LUC reporters and 1µg of control or Spi-B overexpression vectors (pcDNA3.1). Cells were harvested after 24h, after addition of PMA (20ng/ml) and Ionomycin (0.3µM) for the last 6h. Lysates were analyzed with the dual-luciferase reporter assay (Promega). Values are normalized to relative luciferase activity in control transfected cells. Averages ± SD of two experiments are shown.
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