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Blood, Vol. 113, Issue 23, 5911-5919, June 4, 2009
Blimp1 is limiting for transformation in a mouse plasmacytoma model
Blood D'Costa et al.
113: 5911
Supplemental materials for: D’Costa et al
Files in this Data Supplement:
- Figure S1. Serum Ig levels in normal F1 and tumor bearing mice (JPG, 87.5 KB)
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Ig in serum of 4 healthy adult male F1 (C57BL/6 × Balb/c) mice was measured and compared to Ig in serum of plasmacytoma-bearing v-abl–positive mice at the time of euthanasia. “Intact” mice were transgenic and Blimp1+∕+ (solid bars) or Blimp1gfp∕+ (hashed bars). “Reconstituted” mice were generated, as described in the text, by fetal liver transplantation of Rag1−∕− mice with Blimp1+∕+, v-abl+, (solid bars) or Blimp1gfp∕+ (hashed bars) cells. Serum was initially diluted 100-fold for the F1 mice, and 1,000-fold for tumor bearing mice, then serially diluted 2-fold down the ELISA plate. The assay was performed as described in Materials and Methods. Purified mouse monoclonal IgM, IgG1 and IgA purchased from Sigma-Aldrich were used for the quantitative determination of Ig concentrations.
- Figure S2. Phenotypes of additional tumours (JPG, 275 KB)
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(A) Blimp1/GFP+ ASC generated in vitro are heterogenous with respect to Syndecan1 expression. Normal B cells from Blimp1gfp∕+ were cultured for four days under the conditions indicated and analysed for expression of Syndecan1 as described.29 All GFP+ cells in these cultures have been shown to be ASC.29 (B) Corresponding B220 staining of cells from the tumors shown in Fig. 2C. Note all GFP+ cells are B220−. (C) Syndecan1 and B220 staining of tumors from an additional 6 Blimp1+∕+ and 6 Blimp1gfp∕+ mice. Note that while the Sydecan1 levels between some tumours varies significantly, which could be in part due to technical differences of staining and analyses on different days, the B220 levels on B cells within the tumors did not vary significantly. This suggests that the tumors are indeed heterogenous with respect to Syndecan1 expression.
- Figure S3. Expression of Blimp1 alleles and alternative transcripts in normal ASC and tumours (JPG, 104 KB)
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(A) Diagram of the Blimp1 transcription unit, indicating positions of primers specific for the wt allele (a and b alternative transcripts) and the truncated, gfp-reporter (“ko”) allele. The coding sequence is shaded. (B) Cells from BM of normal mice (Blimp1+∕+ and Blimp1gfp∕+), tumours (PCT, plasmacytomas) or in vitro activated B cell cultures were sorted as indicated (markers used are in parentheses). These samples represent normal mature BM plasma cells, in vitro derived B cell blasts and plasmablasts, plasmacytomas and Blimp1-deficient tumors. RNA was extracted and used for semi-quantitative RT-PCR to detect transcripts from the wt and gfp Blimp1 alleles in the cell types listed above. The primary trancript of the wt allele is the a isoform, here detected after 35 PCR cycles (see Materials and Methods). The b isoform is much less abundant, and required 40 cycles for detection. MOPC315, MPC11, P3 and J558 are all established mouse plasmacytoma cell lines. (C) Wild-type Blimp1 mRNA measured by qPCR, using primers that detect only the wt allele (see Methods), in ASC generated in vitro from B6 (Blimp1+∕+, filled bars) and Blimp1gfp∕+, open bars) B cells. Cells were cultured for 4 days as described,29 in LPS alone, or with anti-CD40 plus IL4 and IL5. (D) Western blots of cells from LPS cultures, probed for Blimp1 and actin as a loading control (wt, wild-type; Δ, truncated protein encoded by the Blimp1gfp allele).
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