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Blood, Vol. 113, Issue 11, 2478-2487, March 12, 2009
Expression of sprouty2 inhibits B-cell proliferation and is epigenetically silenced in mouse and human B-cell lymphomas
Blood Frank et al.
113: 2478
Supplemental materials for: Frank et al
SPRY2 promoter-luciferase reporter assay with SssI methyltransferase
Three SPRY2 promoter-luciferase reporter constructs described previously1 were incubated with or without (mock) M. SssI methyltransferase (New England Biolabs, Ipswich, MA) plus S-adenosylmethionine for 1h at 37°C. CpG methylation of each plasmid was determined by BstU1 digestion and agarose gel electrophoresis. Methylated or mock-treated SPRY2 promoter-luciferase reporter constructs (150ng each) were co-transfected with 30ng of pRLCMV-renilla- luciferase control plasmid using FuGENE 6 (Roche, Basel, Switzerland) into HEK293T cells and assayed at 48h for luciferase and renilla-luciferase activity using Dual-Glo (Promega, Madison, WI). Conditional Spry2 knockout mice
Mx1-Cre (Jackson Labs, Bar Harbor, ME) and Spry2fl∕fl mice2 were crossed and injected intraperitoneally at 3–4 weeks of age with sterile 1× PBS, pH 7.4 (control) or 325 µg polyinosinic-polycytidylic acid (poly-dI:dC, P0913, Sigma-Aldrich, St. Louis, MO) to induce Cre-recombinase expression. Mice were sacrificed 2–4 weeks later and the percentage of CD19+/B220+ B cells in the bone marrow and spleen were determined by flow cytometry using CD19-APC (550992, BD Pharmingen, San Jose, CA) and B220-PerCP-Cy5.5 Abs (552771, BD Pharmingen).
Files in this Data Supplement:
- Figure S1. Spry2 is DNA hypermethylated and its expression is repressed in TCL1-tg B-cell lymphomas (JPG, 30.6 KB)
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Spry2 expression in WT (4 pooled spleens) or TCL1-tg B-cell lymphomas was determined by QPCR. Spry2 expression was normalized to 36b4 (Rplp0) expression. DNA methylation status for WT (4 pooled spleens) or 9 TCL1-tg B-cell lymphomas was assessed by MSP. The upper and lower bands correspond to PCR products amplified by primers specific for methylated (M) or unmethylated (U) CpG sites following bisulfite conversion. All tumors were classified using Mouse Models of Human Cancer Consortium (MMHCC) criteria.3 BLL = Burkitt-like lymphoma; DLBCL = diffuse large B-cell lymphoma; MZL = marginal zone B-cell lymphoma.
- Figure S2. DNA hypermethylation of the SPRY2 promoter silences its activity in vitro (JPG, 48.9 KB)
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(A) Three SPRY2 promoter-luciferase reporter constructs, pGL(−1826 to +221)-luc, pGL(-844 to +211)-luc, and pGL(-228 to +221)-luc,1 were treated with SssI methyltransferase or mock treated and digested with the CpG methyl-sensitive restriction enzyme BstU1. L = ladder, M = mock, S = SssI. (B) SPRY2 promoter-luciferase reporter constructs (150ng) were co-transfected with a Renilla-luciferase vector (pRLCMV-luciferase; 30ng) to control for transfection efficiency into HEK293T cells and assayed at 48h for normalized luciferase activity. The activity of the unmethylated pGL(−1826 to +221)-luc construct was set at 100%. Error bars denote s.d. from triplicate experiments.
- Figure S3. Induction of SPRY2 expression is ERK dependent and increases apoptosis in Nalm-6 cells by inhibiting the ERK pathway (JPG, 32 KB)
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(A) SPRY2 protein expression in Nalm-6 cells following stimulation with anti-IgM Ab for the indicated times with (+) or without (−) the MEK1/2 inhibitor, U0126. (B) Western blot for SPRY2 protein expression in Nalm-6 cells stably infected with an empty vector control or a SPRY2-expressing retrovirus. (C) Percentage Annexin V-FITC positive Nalm-6 cells infected with an empty vector control or a SPRY2-expressing retrovirus, and comparison to Nalm-6 cells treated with increasing concentrations of the MEK1/2 inhibitor, U0126. Data is representative of 3 independent experiments.
- Figure S4. ERK1/2 activity is increased in B-cell lymphomas lines with DNA methylated SPRY2 and repressed SPRY2 expression (JPG, 55.1 KB)
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Eight human B-cell lymphoma lines (see Fig. 4A) were unstimulated (upper two panels) or stimulated for 15 min with anti-IgM Ab (lower three panels) and assessed for ERK1/2 phosphorylation and SPRY2 expression by western blot.
- Figure S5. Spry2 null mice have increased CD19+B220+ mature B cells (JPG, 84.6 KB)
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(A) Spry2fl∕fl or Mx1-Cre × Spry2fl∕fl mice were treated with 1× PBS, pH 7.4 or poly-dI:dC, followed by harvest of bone marrow and spleen cells and Spry2 locus-specific PCR from isolated genomic DNA. fl∕fl and null indicate the expected endogenous or Cre-excised band sizes, respectively. (B) CD19/B220 flow cytometry profiles of bone marrow cells from Spry2fl∕fl or Mx1-Cre × Spry2fl∕fl mice injected with poly-dI:dC or 1× PBS, pH 7.4. Data are representative of 16 treated mice and are plotted in aggregate in Fig. 7E.
REFERENCES
1. Ding W, Bellusci S, Shi W, Warburton D. Functional analysis of the human Sprouty2 gene promoter. Gene. 2003;322:175-185.
2. Shim K, Minowada G, Coling DE, Martin GR. Sprouty2, a mouse deafness gene, regulates cell fate decisions in the auditory sensory epithelium by antagonizing FGF signaling. Dev Cell. 2005;8:553-564.
3. Morse HC, 3rd, Anver MR, Fredrickson TN, et al. Bethesda proposals for classification of lymphoid neoplasms in mice. Blood. 2002;100:246-258.
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