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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 S1. Impaired fetal B-cell development after loss of Zfx (JPG, 80.4 KB)
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The analysis of B-cell development in the fetal liver of control and Tie2-Cre+ Zfxflox/y CKO embryos (embryonic day 18.5) and newborns (postnatal day 1). Representative staining profiles are shown, with the B220+ CD43+ Fr. A–C′ pro/pre-B cells highlighted. The fractions of pro/pre B cells indicated are representative of 3–5 embryos/newborns per group.
- Figure S2. Splenic B-cell subsets in Mb1-Cre+ CKO mice (JPG, 58.2 KB)
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The absolute number of splenic B-cell subsets in control and Mb1-Cre+ Zfxflox/y CKO mice at 8–10 wk of age (mean ± SD of 3–4 mice per group). The increase in MZ subset numbers in CKO mice is not statistically significant.
- Figure S3. Zfx deletion in CD19-Cre+ CKO mice (JPG, 350 KB)
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(A) Excision efficiency of Zfx in the indicated sorted B-cell populations in the BM (top panel) and spleen (bottom panel) of CD19-Cre+ Zfxflox/y CKO mice. The presence of Zfxflox or Zfxnull alleles was determined by genomic PCR. (B) The loss of Zfx protein expression in CKO B cells by Western blotting. Splenic B cells were purified from control and CD19-Cre+ CKO mice, and total cell lysates were probed with anti-Zfx polyclonal antibodies.21 (C) The loss of Zfx protein expression in CKO B cells by immunochemistry. Frozen sections of spleens from control and CD19-Cre+ CKO mice were stained with biotin-conjugated anti-IgD followed by AF488-conjugated streptavidin (green), and rabbit polyclonal anti-Zfx followed by AF568-conjugated anti-rabbit IgG (red). Note the absence of nuclear Zfx staining in IgD+ cells from CKO spleens. Bars represent 0.2 mm.
- Figure S4. Normal development of B cells after late B-cell–specific deletion of Zfx (JPG, 372 KB)
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(A) The analysis of Hardy Fractions in the BM of control and CD19-Cre+ Zfxflox/y CKO mice. Representative staining profiles of B220+ CD43+ Fr. A–C′ cells (left panel) and B220+ CD43− Fr. D–F cells (right panel) are shown. The fraction of the following B-cell populations are indicated: left panel, Fr. C late pro-B cells in green and Fr. C′ large pre-B cells in orange (mean ± SD of 4–6 mice per group); right panel, Fr. F recirculating B cells in green (mean ± SD of 11–12 mice per group). (B) The analysis of splenic B-cell populations from control and CD19-Cre+ CKO mice. Representative staining profiles are shown. The fraction of T1 (red), T2 (blue), follicular (green), and marginal zone (orange) are indicated (mean ± SD of 11–12 mice). (C) The absolute number of MZ and FO B-cell subsets in control and CD19-Cre+ CKO mice (mean ± SD of 11–12 mice per group). (D) Normal splenic architecture in CKO mice. Frozen sections of spleens from control and CD19-Cre+ CKO mice were stained with biotin–anti-IgD/AF488-Streptavidin (green) and AF568–anti-IgM (red). Note the normal organization of follicular and marginal zones. Bars represent 1 mm (left panels) and 0.2 mm (right panels).
- Figure S5. Zfx-deficient peripheral B cells are not hyperproliferative in vivo (JPG, 73.7 KB)
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Steady state cell cycle analysis of peripheral B cells in control and CD19-Cre+ CKO mice. Mice were injected i.p. with BrdU 50 min prior to analysis. Representative staining profiles are shown of splenic B220+ B cells and BM B220hi IgDhi recirculating B cells, with cells in G0/G1 (red), S phase (blue), G2/M (green), and sub-G0/G1 (orange) indicated.
Additional supplemental figures can be accessed here.
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