Genes & Development



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Supplemental Table 1. Pathological evaluation of 11-15 month-old Sept4 mice.

The table shows the results obtained from both gross and histopathologic examination of sibling Sept4 wild-type, heterozygous or knock-out homozygous mice from both female and male genders sacrificed at the age of 11-15 months. The percentage of healthy mice was reduced for Sept4+/- mice and Sept4-/- mice compared to their wild-type counterparts. For example, Sept4-/- mice (and to a lesser extent Sept4+/- animals) exhibited an increase in both the incidence and severity of bone marrow dysplasia, lymphoid hyperplasias and leukemias/lymphomas. The spontaneous lymphoma in Sept4-Null mice was very heterogenous and we saw evidence for both B and T-cell lymphoma, and both clonal and polyclonal lymphomas in different animals (see Figure 1 and Supplemental Figure 1). The diagnosis of leukemia and/or lymphoma was based on gross and histological observations that included blast cells covering the tissues and, importantly, disruption of the tissue’s structural organization together with the presence of abnormal mitotic figures. Any case which displayed over-proliferation of lymphocytes but that maintained the structure of the tissue was classified as “hyperplasia”, but never as

“lymphoma”.

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Supplemental Figure 1. Loss of Sept4/ARTS function is associated with hematopoietic malignancies in mice.

(A) Representative bone marrow (BM) FACS profile of an old Sept4-/- mouse which developed BM dysplasia (right panel). This mouse had a BM SSC-FSC profile with a predominant blastoid cell population (gated in red vs regular BM cell populations in black) that does not appear in a normal wild type BM (left panel). The normal BM displays a predominance of differentiated cells. On the contrary, a switch to more mononuclear/immature (blastoid) cells in the bone marrow was confirmed in this Sept4-/- mouse by analyzing fixed tissue sections. (B) Analysis of lymphomas in Sept4 KO mice.

The rearrangements of D and J regions of the immunoglobulin heavy chain genes in different lymphomas was analyzed by PCR. For this purpose, we used nested PCR primers that were specific for the Q52 subset of D-regions and could thus yield four discreet fragments of 1500 bp, 1190 bp, 770 bp and 240 bp, as described in Yu et Thomas-Tikhonenko, 2002 (Oncogene, 21: 1922-1927). Some of the tumors appear to be monoclonal because only the short band appears. However, in other cases we observed several fragments, indicating that these lymphomas are either of polyclonal origin, or that a critical transforming event occurred at a progenitor stage that still allowed Ig rearrangement. (C) Sept4/ARTS is haplo-insufficient for tumor suppression. Relative expression levels of ARTS mRNA were assessed by real-time RT-PCR in samples obtained from Sept4+/- mice that developed lymphomas. Primers used were as described in the main text. Although we found one example where ARTS expression was lost in a tumor of a heterozygous mutant, the majority of Sept4+/- animals retained ARTS expression in the tumor. We conclude that the remaining WT allele has not been lost or silenced, and that Sept4/ARTS displays a moderate haplo-insufficiency for tumor suppression.

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Supplemental Figure 2. Loss of Sept4 function does not lead to increased proliferation of blood cells.

Representative Propidium Iodide (PI) cell cycle profiles are shown for activated B (A), activated T (B) cells and thymocytes (C). The percentage of cells within each phase of the cell cycle is depicted next to the corresponding plot. (D) Table summarizing the percentage values of each cellular population in each phase of the cell cycle (n=2 to 10; paired t-test; mean ± SEM). This analysis revealed no evidence that the loss of Sept4 function causes increased tumor formation through increased cell proliferation. (E) Histogram displaying the percentage of Sept4-/- or WT-derived T cells activated with concanavalin A that underwent 1-6 cell divisions measured by CFSE dilution. For cells that divided multiple times (more than 4 times), loss of Sept4 function caused slightly reduced growth. (F) FACS profiles showing CFSE fluorescence of unstimulated (blue line) or concanavalin A and IL-2-activated T cells (red line). The number of cells that did not divide at all (right peak) or divided a small number of times was slightly higher for Sept4-/- T cells.

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Supplemental Figure 3. Young Sept4-/- mice have normal cellular composition of the spleen and thymus.

A cell suspension was prepared from the spleen (upper charts) and thymus (lower charts) of 6-13 week-old wild type and Sept4-deficient mice and their cellular distribution analyzed by staining specific surface receptors and FACS. Similar distribution of B cells (CD19pos) and T cells (CD90pos) was observed for the spleen of wild type and knock-out mice. In the same way, the percentage of mature (CD4pos or CD8pos only) and immature thymocytes (double CD4/CD8pos) showed no differences. Each dot indicates the value obtained from a single mouse and the line shows the mean for each group (paired t-test).

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Supplemental Figure 4. The loss of Sept4 function does not cause increased proliferation of hematopoietic stem and progenitor cells.

(A) Representative cell cycle profiles of B cell progenitors (above) and LSK cells (below) of indicated genotypes. The percentage of cells within each phase of the cell cycle is depicted next to the corresponding plot. (B) Table summarizing the percentage values of B-cell progenitors and LSK cells in each phase of the cell cycle (n=7; paired t-test; mean ± SEM). No significant differences were observed in the PI cell cycle profile between the wild-type and Sept4-/- cells. (C) Histogram showing the percentage of LSK in the bone marrow that incorporates BrdU over the course of 24 hours. Values are mean ± SEM, n=2. These results suggest that an extra-proliferating state should not be the mechanism to explain the increase in stem/progenitor cells of Sept4-/- mice.

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Supplemental Figure 5. ARTS mRNA is the most abundant Sept4 isoform in hematopoietic stem and progenitor cells.

Relative expression levels of mRNA from different Sept4 isoforms were assessed by real-time RT-PCR in LSK (upper chart) and B cell progenitors (sorted as B220posIgMneg; lower chart). Whereas the “traditional” septin isoforms, including H5, are prevalent in various differentiated cell types and mature tissues (Larisch et al., 2000, Kissel et al., 2005), ARTS is expressed at high levels in LSK cells and B-cell progenitors.

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Supplemental Figure 6. Loss of Sept4 function accelerates lymphoproliferation in the Eμ-myc model.

(A) Cell blood counts from Eμ-myc mice at the age of 5 and 8 weeks. Blood samples were obtained retro-orbitally and analyzed in a regular blood cytometer. Increased numbers of circulating white blood cells were observed in Sept4-/- compared to WT mice. (WBC, white blood cells; RBC, red blood cells; HGB, hemoglobin; HCT, hematocrit). (B) Analyses of lymph organ cellularity showed significantly higher numbers of cells in the BM of Eμ-myc/Sept4-/- compared to Eμ-myc/Sept4+/+ mice (4-5 week-old; paired t-test). A cellular suspension was obtained from the spleen, thymus and bone marrow of the mice and the total number of cells was calculated with a regular haemocytometer. Although the number of cells was similar for the spleen and thymus of Eμ-myc/Sept4-/- and wild-type mice, there was a significant increase in the total cellularity of the bone marrow when Sept4 is loss indicating the development of leukemia/lymphoma. Each dot corresponds to one mouse and the line indicates the mean value. (C) Loss of Sept4 function does not inhibit c-myc induced apoptosis in lymphocytes. B-cells expressing Eμ-myc in either a WT or Sept4-Null background were isolated and apoptosis was measured by staining for both active caspase-3 (upper panel) and AnnexinV-PI (lower panel). The response was evaluated both with and without the addition of different apoptotic inducers. No differences were observed for c-myc expressing B cells from WT and Sept4-Null animals (n= 4, mean ± SEM is represented, paired t-test). (D) Analysis of tumors in Eµ-myc/Sept4-/- mice. Cell suspensions were obtained from the enlarged lymph node of a Eµ-myc/Sept4-/- mouse and stained using surface receptor markers. Cells were then analyzed by FACS. The dotted line represents were the low-high level of expression is set up. Cells highly express B220 and CD19 markers. They also express lower levels of the surface markers CD90, CD43 and CD4 and (although at low levels) Sca-1 and c-kit receptors. Most cells were negative for IgM, IgD and CD8. Several different mice were analyzed and yielded similar results (data not shown). These results indicate that tumors in Eµ-myc/Sept4-/- mice are primarily composed of primitive cells.

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Supplemental Figure 7. Loss of Sept4 function does not affect mitochondrial ultrastructure.

Transmission electro-microscopy images showing the structure of mitochondria in Linneg cells isolated from the bone marrow of Sept4-/- mice compared to their WT counterparts. No differences in the mitochondrial ultrastructure were observed between WT and Sept4-Null in Linneg cells as well as various other cell types (see also Kissel, 2005, supplementary information).

Supplemental Materials and Methods

Cell cycle by Propidium Iodide staining

Lymphocytes were fixed in ice-cold 70% ethanol for at least 2 hours. The ethanol suspended cells were centrifuged at 200 g for 5 minutes and then washed with 5 ml PBS. Cell pellets were suspended in 1 ml PI/Triton X-100 staining solution (10 ml of 0.1% Triton X-100 in PBS, 2 mg DNase-free RNase A and 200 µl of 1 mg/ml Propidium Iodide) for 30 minutes at room temperature and analyzed by flow cytometry.

CFSE staining

T-cells isolated from the spleen were incubated for 10 minutes in RPMI-1640 plain medium with 5µM carboxyfluorescein diacetate succinimidyl ester (CFSE; Molecular Probes) at 37°C. The reaction was stopped with cold RPMI-1640 complete media and the cells cultured at a concentration of 1.2x106 cell/ml for 3 days with concanavalin A (2µg/ml) and IL-2 (10ng/ml) to induce proliferation and activation. The T-cell proliferation was determined by the decline in CFSE fluorescence by FACS analysis. CFSE-stained T-cells cultured without Concanavalin A and IL-2 were used as non-activated control.

BrdU stain

The APC-BrdU Flow Kit (BD Biosciences) was used following a single intraperitoneal injection of BrdU (1 mg per 6 g of body weight) and an additional administration of 1 mg/ml of BrdU (Sigma) mixed to drinking water for 24h. The BrdU staining was performed following the manufacturer’s instructions.

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