E Michalak et al. Supplementary Material Supplementary Materials and Methods Real time qRT-PCR analysis primer sequences. qRTPCR was performed using the following forward and reverse primers (a kind gift from Drs DCS Huang and KJ Campbell) gene sense (5’ – 3’) antisense (5’ – 3’) a1 GTCATACTTGGATGACTTTCACGTG ATTCTCCTGTGTTATTCATTATGAATTCTG bad AGTATGTTCCAGATCCCAGAGTTTG CCTTGAAGGAACCCTCAAACTCATC bcl-2 TTATAAGCTGTCACAGAGGGGCTAC GAACTCAAAGAAGGCCACAATCCTC bcl-xl TGGAGTCAGTTTAGTGATGTCGAAG AGTTTACTCCATCCCGAAAGAGTTC bim GAGTTGTGACAAGTCAACACAAACC GAAGATAAAGCGTAACAGTTGTAAGATA mcl-1 GAGGAGGAAGAGGACGACCTATACC AGTTTCTGCTAATGGTTCGATGAAG puma ATGCCTGCCTCACCTTCATCT AGCACAGGATTCACAGTCTGGA noxa ACTGTGGTTCTGGCGCAGAT TTGAGCACACTCGTCCTTCAA c-myc CAAATCCTGTACCTCGTCCGATTC CTTCTTGCTCTTCTTCAGAGTCGC Supplementary Figure Legends Supplementary Figure 1 Loss of Puma affects some B lymphoid cell subsets in the lymph nodes and spleen of E-myc mice. Total cellularity and cell subset composition of pre-neoplastic 5-6 week old mice of the indicated genotypes. (A) Numbers of preB, virgin and mature B cells in the lymph nodes (axillary, brachial, inguinal pairs) were determined by immunofluorescent staining with surface marker-specific monoclonal antibodies and flow cytometric analysis. Differences between E-myc Michalak et al transgenic and non-transgenic mice were significant for all puma genotypes for pre-B cells, as were comparisons of virgin B cells for wt vs E-myc/puma-/- mice and for the decrease in mature B cells for wt vs E-myc/puma+/- and wt vs E-myc/puma-/- mice (P < 0.005). For comparisons of E-myc transgenic mice of the different puma genotypes, all statistically significant differences are indicated. (B) Similar analysis of the spleen revealed differences between E-myc and non-transgenic mice of all puma genotypes (P < 0.005) for pre-B cells and mature B cells and for total cells and virgin B cells for wt vs E-myc/puma-/- mice. For comparisons of E-myc transgenic mice of the different puma genotypes, all statistically significant differences are shown. Values represent means ± SEM from 5-8 mice of each genotype. * P < 0.05, *** P < 0.005. Supplementary Figure 2 Impact of Puma loss on survival of E-myc pro-B cells and sIg+ B cells in culture. (A) Pro-B cells or (B) virgin/mature B cells sorted from the bone marrow of 5-6 week old healthy wt mice or E-myc mice of the indicated puma genotypes were cultured in simple medium (no cytokines) or treated with etoposide (1 g/mL) for the indicated times. For all experiments cell viability was determined by FACS analysis as the percentage of Annexin V-PI- cells. Values represent means ± SD of cells from 3-6 mice of each genotype. No statistically significant differences were noted between transgenic genotypes. Similar results were obtained with virgin (sIgMhisIgDlo) and mature (sIgMlosIgDhi) B cells from the spleen (data not shown). Supplementary Figure 3 Representative immunophenotyping FACS plots of lymphomas from E-myc and E-myc/puma-/- mice. Cell suspensions were prepared from primary lymphomas and flow cytometric analyses performed after 2 Michalak et al immunofluorescent staining with surface marker-specific monoclonal antibodies. Lymphomas were classified as (A) B220+sIg- pro-B/pre-B, (B) B220+sIgM+sIgDlo B cell or (C) “mixed” pre-B/B cell if distinct sIg- and sIg+ B lymphoma populations were present. Supplementary Figure 4 Loss of Puma affects the numbers of B lymphoid cells in E-myc mice in the absence of Noxa. (A) White blood cell counts (means ± SEM) of pre-neoplastic 4 week-old mice of the indicated noxa genotypes. Cell counts for Emyc mice of each of the noxa genotypes did not differ significantly, but the differences from their non-transgenic counterparts were highly significant (P < 0.0001). (B) Spleen weights (means ± SEM) of pre-neoplastic 5-6 week-old mice of the indicated noxa genotypes. Differences between non-transgenic and E-myc or E-myc/noxa-/- mice were significant (P < 0.001). Spleen weights of E-myc and Emyc/noxa-/- mice were similar (P = 0.76). (C) B lymphoid cell subset composition of lymph nodes, peripheral blood and spleen from pre-neoplastic 5-6 week-old mice of the indicated genotypes. Total cellularity and cell subset composition were determined as described in Supplementary Figure 1. For lymph nodes, statistically significant differences in addition to those indicated were noted for total cells and preB cells for E-myc vs E-myc/noxa-/-puma+/- mice. For peripheral blood, statistically significant differences in addition to those indicated were noted for all comparisons except between E-myc vs E-myc/noxa-/- mice. For spleen, statistically significant differences in addition to those indicated were noted for total cells, virgin B cells and mature B cells for E-myc vs E-myc/noxa-/-puma+/- mice and for total cells, pre-B cells and virgin B cells for E-myc/noxa-/- and E-myc/noxa-/-puma-/- mice 3 Michalak et al (P < 0.005). Values in (C) represent means ± SEM from 3-7 mice of each genotype. * P < 0.05, ** P < 0.01, *** P < 0.005. Supplementary Figure 5 Loss of Noxa does not enhance the survival of E-myc transgenic B lymphoid cells in culture. Pro-B cells (A), pre-B cells (B) or virgin and mature B cells (C) were sorted from the bone marrow of 5-6 week old healthy wt mice or E-myc mice of the indicated noxa genotypes and cultured in simple medium (no added cytokines) or treated with etoposide (1 g/mL) for the indicated times. Cell viability was determined by FACS analysis as the percentage of Annexin V-PI- cells. Data points represent means ± SEM of cells from 3-6 mice of each genotype. Statistically significant differences noted: E-myc vs E-myc/noxa-/-puma-/- and Emyc/noxa-/- vs E-myc/noxa-/-puma-/-, P < 0.5 for pro-B cells and virgin/mature B cells at 4 hours post etoposide only. Supplementary Figure 6 Loss of Noxa does not accelerate lymphomagenesis in Emyc mice. Kaplan-Meier analysis of tumour-free survival of mice of the indicated genotypes. Differences in tumour onset between E-myc and E-myc/noxa+/- or Emyc/noxa-/- mice were not significant (P = 0.18 and P = 0.13, respectively). Supplementary Figure 7 Western blot analysis of p19Arf expression levels in lymphomas from E-myc mice of the various puma and noxa genotypes. Western blot analysis of p19Arf and -actin (loading control) on protein isolated from lymphoma suspensions from E-myc mice of the indicated puma and noxa genotype. Lysates from p53-/- mouse embryonic fibroblasts (MEF) or a tumour (E-myc tumours known 4 Michalak et al to be positive for p19Arf) were included as positive controls for p19Arf overexpression. Protein size standards in kDa are indicated on the left-hand side. Supplementary Figure 8 Assessment of p53 status and function in E-myc lymphomas. (A) Genomic PCR of the Ink4A/arf locus revealed that no E-myc/puma/- lymphoma was observed to have a deletion of this gene. Controls: E-myc lymphoma cell line known to contain a deletion in the Ink4A/arf locus; spleen from an Ink4A/arf deficient mouse; E-myc/p53-/- lymphoma known to retain the Ink4A/arf locus; E-myc lymphoma known to retain the Ink4A/arf locus. (B) Assessment of p53 status and function in cohorts of E-myc lymphomas with or without Puma. *Lymphoma cell lines displayed p53 deficiency-like resistance following in vitro exposure to etoposide. ND = Not Done. 5