Overexpression of MYC in HSC induces both myeloid and lymphoid leukemia.

<p>Flow cytometry analysis of bone marrow and spleen cells from two individual moribund Mock/MYC mice (indicated with # in the left). In the middle, percentage of GFP⁺YFP⁺ (Mock/MYC expressing) cells or GFP⁻YFP⁺ (MYC expressing) cells are indicated. These cells were further characterized with anti-CD11b, anti-Gr1, anti-CD4, anti-CD8, anti-CD19, anti-IgM anti-CD71 and anti-Ter119. The percentage of cells in each quadrant is indicated.</p

BCL-X_L and BCL-2 but not FLIP_L accelerate Myc-induced hematopoietic tumorigenesis.

<p>Kaplan-Meier survival analysis of mice transplanted with HSC over-expressing (A) Mock-GFP/MYC-YFP in DBA/2, (B) BCL-X_L -GFP/MYC-YFP in DBA/2, (C) FLIP_L-GFP/MYC-YFP in DBA/2, (D) BCL-X_L-GFP/MYC-YFP in BALB/c and (E) BCL-2-GFP/MYC-YFP in BALB/c. Mice were monitored daily for tumors or signs of paralysis and killed if showing signs of sickness. The percentage of mice surviving at daily intervals is shown.</p

Blast formation of Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP or BCL-X_L-GFP/MYC-YFP bone marrow and spleen cells.

<p>(A) Forward light scatter of CD11b⁺Gr1⁺ bone marrow cells from Mock-GFP/Mock-YFP, Mock-GFP/MYC-YFP and BCL-X_L-GFP/MYC-YFP recipient mice 2 weeks after transplantation. Grey thick lines indicate GFP⁻YFP⁺ cells, thick black lines indicate GFP⁺YFP⁻ cells and thin lines indicate GFP⁺YFP⁺ cells. (B) Difference in mean fluorescence intensity (Δmfi) in forward scatter of CD11b⁺Gr1⁺ (left), CD19⁺IgM⁻ (middle) and CD19⁺IgM⁺ (right) cells in bone marrow (top) and spleen (bottom) between GFP⁻YFP⁻ (non-transduced cells) and GFP⁻YFP⁺ (black bars), GFP⁺YFP⁺ (striped bars) or GFP⁺YFP⁻ (white bars) cells are shown. Values indicate means of three individual mice and error bars indicate 1 SD.</p

Over-expression of BCL-X_L and BCL-2 accelerate Myc-induced splenomegaly whereas co-expression of FLIP_L does not influence MYC-induced splenomegaly.

<p>Spleen weight of animals transplanted with HSC expressing (A) Mock-GFP/Mock-YFP into DBA/2 mice, (B) Mock-GFP/MYC-YFP into DBA/2 mice, (C) FLIP_L-GFP/MYC-YFP into DBA/2 mice, (D) BCL-X_L-GFP/MYC-YFP into DBA2 mice, (E) BCL-X_L-GFP/MYC-YFP into BALB/c mice and (F) BCL-2-GFP/MYC-YFP into BALB/c. Spleens of mice at control time points (7 days, 14 days, 35 days and 49 days after transplantation) and spleens of all moribund mice were weighed. Each dot represents one mouse.</p

Overexpression of BCL-X_L-GFP/MYC-YFP induces AML.

<p>Flow cytometry analysis of bone marrow (top), spleen (middle) and liver (bottom) cells from one representative moribund BCL-X_L-GFP/MYC-YFP mouse. Single cell suspensions from femoral bone marrow, spleen and liver were stained with anti-CD11b and anti-Gr1. The percentage of dominating cells is indicated.</p

Tumor phenotype in Mock/MYC, FLIP_L/MYC, BCL-X_L/MYC and BCL-2/MYC recipient mice.

<p>Summary of flow cytometry analysis of spleen cells from moribund mice transplanted with HSC expressing (A) Mock-GFP/MYC-YFP into DBA/2 mice, (B) FLIP_L-GFP/MYC-YFP into DBA/2 mice, (C) BCL-X_L-GFP/MYC-YFP into DBA2 mice, (D) BCL-X_L-GFP/MYC-YFP into BALB/c mice and (E) BCL-2-GFP/MYC-YFP into BALB/c mice. Left panel specify tumors expressing MYC-YFP only and right panel specify cells expressing MYC-YFP together with either Mock-GFP (A), FLIP_L-GFP (B), BCL-X_L-GFP (C and D) or BCL-2-GFP (E). Filled box indicate tumor phenotype. M indicates tumors of myeloid lineage, DP indicate CD4⁺CD8⁺ lymphoid tumors, 4 indicate CD4⁺ lymphoid tumors and 8 indicate CD8⁺ lymphoid tumors. Numbers correspond to identity of individual animal.</p

Statistical analysis of survival data after transplantation of hematopoietic stem cells expressing MYC, BCL-X_L, BCL-2 and/or FLIP_L.

<p>Statistical analysis of the survival data presented in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0031366#pone-0031366-g002" target="_blank">Figure 2</a>.</p>*<p>Log-rank (Mantel-Cox) test. NS = non significant. S = significant. P-value indicated below.</p

Expression of the various combinations of genes in Lin⁻ cells prior to transplantation.

<p>FACS analysis of expression of YFP and GFP in HSC prior to transplantation in HSC derived from (A) DBA/2 mice transduced with Mock-GFP and Mock-YFP, (B) DBA/2 mice transduced with Mock-GFP and MYC-YFP, (C) DBA/2 mice transduced with BCL-X_L-GFP and MYC-YFP, (D) DBA/2 mice transduced with FLIP_L-GFP and MYC-YFP, (E) BALB/c mice transduced with BCL-X_L-GFP or MYC-YFP and (F) BALB/c mice transduced with BCL-2-GFP and MYC-YFP. The percentage of cells expressing the genes in each quadrant, gated on PI negative cells, is indicated.</p

Expression of SOXC genes in MCL cell lines.

<p>Expression of SOX4 (A), SOX12 (B) and SOX11 (C) in the MCL cell lines Granta 519, JeKo, Rec1 and JVM2 by quantitative RT- PCR. Granta 519 express high levels of SOX11 mRNA but low levels of the other SOXC genes while no SOX11 mRNA expression was detected in JVM2. (D) WB showed that Granta 519, JeKo and Rec1, but not JVM2, express SOX11 protein.</p

SOX11 silencing significantly reduces TUBB3 expression in the MCL cell line Granta 519.

<p>(A) Expression of SOX11 was evaluated after transfection with either 100 pmol SOX11 siRNA or control siRNA. The SOX11 mRNA level was measured by quantitative RT-PCR 20 hours after electroporation. Error bars show standard deviation of 4 independent experiments, ***p<0.001. (B) Western blots show downregulation of SOX11 protein in Granta 519 after SOX11 siRNA knockdown but not in cells treated with control siRNA or in untransfected cells. (C) Depletion of SOX11 protein significantly reduces TUBB3 expression. Granta 519 cells were transfected with SOX11 siRNA and TUBB3 expression was investigated by quantitative RT-PCR after 20 hours. ***P<0.001.</p