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Blood, Vol. 112, Issue 13, 5063-5073, December 15, 2008
NF- B1 and c-Rel cooperate to promote the survival of TLR4-activated B cells by neutralizing Bim via distinct mechanisms
Blood Banerjee et al.
112: 5063
Supplemental materials for: Banerjee et al
Macrophage cultures. wt and nfkb1−/− bone marrow-derived macrophages were generated using L-cell conditioned medium containing M-CSF as previously described.11 Equivalent numbers of macrophages of both genotypes were seeded into 96 well flat-bottom plates in RPMI1640, 10% fetal bovine serum containing 10% L-cell conditioned medium, 50 µM 2-mercaptoethanol and either left untreated or stimulated with 20 µg/ml LPS. Measuring macrophage viability. To assess macrophage viability, cultures were first subjected to centrifugation (400 ×g/5 min), the pelleted cells washed and the centrifugation step repeated. Cells were then incubated with 1 µM calcein and 1 µM of ethidium homodimer (in KDS Balanced Salt Solution) for 20 min at 37°C. Cells were then examined under a fluorescence microscope using a 490 nm excitation wavelength filter. Viable cells (green) and apoptotic cells (red) were counted in a defined field. At least 200 cells/well were counted and in each experiment there were duplicate or triplicate cultures.
Files in this Data Supplement:
- Table S1 (PDF, 32.7 KB) -
Representative spleen cellularities (×106) for wild-type, nfκb1−/−, bim−/−, nfκb1−/−bim−/−, bcl-2T, and nfκb1−/−bcl-2T mice.
- Figure S1. Bcl-2 transgene expression in wt and nfkb1−/− B cells: Western blot analysis of endogenous mouse Bcl-2 (mBcl-2) and transgenic human (JPG, 26.6 KB)
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Bcl-2 (hBcl-2) expression in wt, bcl-2T, nfkb1−/− and nfkb1−/−bcl-2T B cells prior to and following LPS stimulation (18 hrs). Actin loading control is shown.
- Figure S2. Profiles of follicular and marginal zone B-cell populations in nfκb1−/−bim−/− mice (JPG, 178 KB)
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Spleens from wt, nfκb1−/−, bim−/−, nfκb1−/−bim−/−, bcl-2T, and nfκb1−/−bcl-2T mice were stained with cell surface marker specific antibodies and FACS analysis performed to assess their content of marginal zone (IgM+CD23−CD21+) and follicular (IgM+CD23+CD21+) B cells. Representative FACS plots show CD21 versus IgM staining profiles of CD23+ or CD23− gated populations. The total number of each B-cell population is shown for mice of each genotype. The data shown are representative of at least 3 mice per genotype and three independent experiments.
- Figure S3 (JPG, 40.5 KB)
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(A) 4-HT induces ERK activation in the WEHI 231 B lymphoma cell line expressing a c-Raf:ER fusion protein. WEHI 231 B cells infected with the retrovirus pMy-IRES-GFP-Raf:ER were untreated or treated with 4-HT over a 2 hr period. Total cell lysates were subjected to Western blotting using antibodies specific for p-ERK, Bim and ERK. (B) The proliferation of LPS stimulated B cells is blocked by constitutive Raf:ER activation. wt, nfkb1−/−, Raf:ER, and nfkb1−/−Raf:ER B cells were activated in culture for 48 hrs with LPS, 4-HT or LPS and 4-HT. B-cell proliferation was assessed by 3H-thymidine incorporation. The results represent means ± SEM of 3 independent experiments, each performed in triplicate. Incorporated 3H-thymidine of less than 103 cpm is indicated as <103 (lanes 5–8).
- Figure S4 (JPG, 41.2 KB)
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(A) Time course of LPS induced Bim phosphorylation in B cells. Lysates isolated from wt B cells prior to or following LPS stimulation for 1, 1.25, 2, 4 and 6 hrs were fractionated on 2D-gels and subjected to Western blot analysis using Bim specific antibodies. The symbols + and – refer to the anode and cathode respectively. (B) The 4-HT induced phosphorylation of Bim in B cells expressing Raf:ER is blocked by PD98059. WEHI 231 B cell infected with the retrovirus pMy-IRES-GFP-Raf:ER were either untreated, or treated for 2hrs with PD98059, 4-HT or PD98059 plus 4-HT. Total cell lysates fractionated on 2D-gels were subjected to Western blot analysis using Bim specific antibodies. The symbols + and – refer to the anode and cathode respectively.
- Figure S5. LPS does not induce increased apoptosis in nfkb1−/− macrophages (JPG, 14.7 KB)
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Equivalent numbers of wt and nfkb1−/− bone marrow derived macrophages were left untreated or stimulated with LPS over a 48 hr period. Cell viability at T0, T24 and T48 hrs was determined by microscopic examination of cells stained with calcein and ethidium. The results represent the mean ± SEM of 3 independent experiments.
- Figure S6. The increased spontaneous and LPS induced death of nfkb1−/− B cells is not influenced by the absence of the BH3-only pro-apoptotic protein Bad (JPG, 54.5 KB)
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wt, nfkb1−/−, bad−/−, and nfkb1−/−bad−/− B cells were cultured in simple medium or stimulated with LPS over 48 hr. The viability of B cells from mice of all genotypes was >98% prior to culture. (A) Cells cultured in the absence of mitogenic activation were harvested after 24 and 48 hrs and the frequency of apoptotic cells determined. (B) B cells were stimulated with LPS for 24 and 48 hrs, after which levels of cell death were determined. Both sets of results represent the mean ± SEM of 3 independent experiments.
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