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Blood, Vol. 113, Issue 23, 5857-5867, June 4, 2009
Transcription factor Zfx controls BCR-induced proliferation and survival of B lymphocytes
Blood Arenzana et al.
113: 5857
Supplemental materials for: Arenzana et al
Files in this Data Supplement:
- Figure S6. Response to T-independent type 2 antigen is minimally affected in Zfx-deficient mice (JPG, 105 KB)
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Humoral response to T-independent antigen in control (filled circle) and CD19-Cre+ CKO (open diamond) mice. Mice were immunized with NP-Ficoll and sera taken at the indicated time points. Relative anti-NP IgM, IgG1, and IgG3 are shown as determined by NP-specific ELISA. Each symbol represents an individual mouse and the bar shows the mean. (n.d., not detectable; **P 0.01).
- Figure S7. Initial delay in antibody response, but normal memory response to T-dependent antigen in Zfx-deficient mice (JPG, 120 KB)
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Humoral response to T-dependent antigen in control (filled circle) and CD19-Cre+ CKO (open diamond) mice. Mice were immunized with NP-KLH in alum then given a secondary immunization (arrow) on day 42, with sera taken at the indicated time points. Relative anti-NP IgM, IgG1, IgG2a, and IgG2b are shown as determined by NP-specific ELISA. Each symbol represents an individual mouse and the bar shows the mean. (n.d., not detectable; *P < 0.05, **P 0.01).
- Figure S8. Impaired survival of Zfx-deficient B cells upon BCR stimulation (JPG, 66.9 KB)
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(A) Apoptosis of B cells from control and CD19-Cre+ CKO mice. Resting B cells were cultured in vitro with indicated condition for 24 hrs. The fractions of Annexin V+ 7-AAD− apoptotic cells and Annexin V+ 7-AAD+ dead cells are shown (mean ± SD of 3 mice per group). *P < 0.05, **P 0.01, ***P < 0.001. (B) Proliferation of IgM-stimulated B cells from control and CD19-Cre+ CKO mice. The CFSE dilution data from Fig. 5C are shown on the same scale without normalization. Note that the CFSEbright peak is reduced in CKO B cells, particularly after anti-IgM and LPS stimulation. This suggests increased cell death of non-proliferating cells, consistent with the data in panel A. On the other hand, the amount and cycle distribution of proliferating cells are not reduced after anti-CD40 and LPS stimulation.
- Figure S9. Normal BAFF responsiveness of Zfx-deficient B cells (JPG, 189 KB)
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(A) BAFF-mediated survival of B cells from control and CD19-Cre+ CKO mice. Enriched B cells were cultured in vitro in the absence or presence of 200 ng/ml recombinant BAFF for 2 or 4 days. Shown are representative staining profiles of two independent experiments, with the fraction of Annexin V− 7-AAD− viable cells indicated. Note the increased fraction of viable cells in control and CKO B cells in the presence of BAFF. (B) Sorted splenic T1 cells were cultured with BAFF for 4 days, and analyzed as above. Similar results were obtained with T2 cells (not shown).
- Figure S10. Proximal BCR signaling events in Zfx-deficient B cells (JPG, 211 KB)
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The analysis of downstream effectors in BCR-induced B cells from control and CD19Cre/+ Zfxflox/y CKO mice. Whole cell lysates were prepared from resting B cells stimulated with α-IgM for the indicated time points and analyzed by Western blotting with indicated antibodies. Data are representative of two or three independent experiments.
- Figure S11. Unfolded protein response is not induced in BCR-stimulated Zfx-deficient B cells (JPG, 54.2 KB)
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The analysis of spliced and unspliced XBP-1 in BCR-stimulated B cells from control and CD19Cre/+ Zfxflox/y CKO mice. Resting B cells were stimulated with α-IgM or LPS for the indicated time points. Total cell RNA was reverse-transcribed and assayed by RT-PCR. Primers (5′- ACACGCTTGGGAATGGACAC-3′ and 5′- CCATGGGAAGATGTTCTGGG-3′) surrounding spliced area of Xbp-1 were used for PCR amplification as described (Iwakoshi, N.N., Lee, A.H., Vallabhajosyula, P., Otipoby, K.L., Rajewsky, K., and Glimcher, L.H. (2003). Plasma cell differentiation and the unfolded protein response intersect at the transcription factor XBP-1. Nat Immunol 4, 321–329).
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