Figure 2

Striking up-regulation of MCC in TRAF3^-/-mouse B lymphomas but not in premalignant TRAF3^-/-B lymphocytes. (A) Western blot analysis of the MCC protein. Total cellular proteins were prepared from purified LMC splenic B cells or splenic B lymphomas (spl) or ascites (asc) of different individual B-TRAF3^−/− mice. (B) Regulation of the expression of MCC and Zcwpw1 in response to B cell stimuli. LMC and premalignant (young) TRAF3^-/- splenic B cells were cultured ex vivo in the absence or presence of stimuli of B cell proliferation, differentiation and activation for 6 or 24 hours. B cell stimuli examined include: 2 μg/ml anti-CD40, 20 μg/ml LPS, 1 μg/ml anti-BCR, and 100 nM CpG2084, alone or in combination. RNA samples of TRAF3^-/- B lymphomas (mouse ID: 7060–8) were used as positive control of the MCC and Zcwpw1 transcripts in Taqman assays. (C) Normal copy number of the MCC gene in TRAF3^-/- mouse B lymphomas. Copy number of the mouse MCC gene in genomic DNA samples prepared from LMC splenocytes or TRAF3^−/− B lymphomas was determined using the TaqMan Copy Number Assay kit. Mouse ID was indicated in the figure. (D) Histone modifications of the promoter region of the MCC and Diras2 genes. Chromatin was prepared from LMC and young TRAF3^-/- splenic B cells, or TRAF3^-/- B lymphomas. Fragmented chromatin was immunoprecipitated using antibodies specific for histone marks (H3K27me3 or H3K9/14ac) or non-specific rabbit Ig. Immunoprecipitated DNA was quantified by qPCR using primer pairs specific for the promoter region of the MCC and Diras2 genes, respectively. Quantity of immunoprecipitated DNA is presented as percentage of input DNA in graphs. All graphs (B-D) depict the results of three independent experiments with duplicate reactions in each experiment (mean ± S.D.). *P < 0.05, and **P < 0.0001 by Student’s t test.