This file contains supplementary methods and Figure legends for three supplementary Figures (Fig. S1, Fig. S2, Fig. S3).

Supplementary Methods

Mice

Genotyping was performed by PCR analysis of tail DNA using the following primers: mycS, 5`-CGGACACACAACGTCTTGGA-3´, and mycAS, 5`-CTCTCACGAGAGATTCCA GC-3´.

Constructs, Transfection and Retroviral Infection

Human MCL-1 was amplified by PCR and cloned into the pMIG retroviral vector using the following primers: hMCL-1S, 5´-CGCGGATCCACCATGTTTGGCCTCAAAAGAAACGC-3`, and hMCL-1AS, 5`-CCGGAATTCCGGCTATCTTATTAGATATGCCAAACCA-3. The MCL-1 mutant S159A was generated using the Quikchange Kit (Agilent) following primers: hMCl-1S, 5`-ACGGACGGGGCACTACCCTCGA-3`, and hMCL-1AS, 5`-TCGAGGGTAGTGCCCCGTCCGT-3. 293T cells were infected with Hit60, VsVg and pMIG constructs using superfect (Qiagen) according to manufacturers protocol. After 16h, sodium butyrate was added (5mM) for 8h. Retrovirus-containing supernatant was harvested after 12h and 24h, respectively. C57BL/NCrl or Eµ-myc mouse BM was mobilized with 5-Fluoruracil (150mg/kg), isolated 3 days after injection and taken in culture with IMDM containing IL-3 (10ng/ml), IL-6 (10ng/ml) and mSCF (50ng/ml). After 12h in culture, the BM cell culture was infected with the respective virus supernatants, which was repeated after 10h. The infection level (GFP) of BM cells was monitored after 10h by FACS and 5x105 infected BM cells were engrafted in 6-8 weeks old irradiated (9.5Gy) female C57BL/NCrl mice.

ADVIA hematology analyzer and Flow cytometric analysis

Peripheral blood was analyzed using the ADVIA hematology analyzer and by FACS. Hematopoietic organs were isolated and single-cell suspensions of bone marrow, lymph nodes, spleen and thymus prepared. Cells were counted with the Z1 Coulter Counter and 1x106 cells stained for certain cell populations using specific monoclonal antibodies: 500A2, anti-CD3e; H129.19, anti-CD4; 53-6.7, anti-CD8a; RA3-6B2, anti-CD45R-B220 (Becton Dickinson); M1/70, anti-CD11b; 1B11, anti-CD43; RMM-1, anti-IgM; 11-26c.2a, anti-IgD; D7, anti-Sca-1; RB6-8C5, anti-Gr-1 (BioLegend); 3C1, anti-c-Kit (Milteny Biotec); hematopoietic lineage biotin panel (CD3e, CD11b, CD45R-B220, Ter-119, and Ly6G-Gr-1) (eBioscience).

Histology

Tissues were fixed in 4% buffered formalin, decalcified in a mixture of 10 % ethylene-diamine-tetraacetic acid disodium salt (EDTA, Serva, Cat. No. 11280.02) and 3.3 % tris-(hydroxymethyl) aminomethane (THAM, AppliChem, Cat. No. A1086,1000) in dd H2O at pH: 7.0 – 7.2 and paraffin embedded. Histological examination of all the specimens was performed using hematoxylin-eosin (H&E) staining.

Proliferation using RTCA analyzing system

HeLa cells were plated (5000/ well) onto 96-well E-plates and recorded every 15 min for 49 h over time.

Western blotting

Immunoblotting was performed using antibodies raised angainst MCL-1 (S-19, sc-819, Santa Cruz) and GFP (#2255, Cell Signaling Technology).

Real-time PCR

Relative expression of MCL-1 was determined by real-time PCR in comparison to the L32 housekeeping gene as described before6. Primers sequences are as follows: mL32F: TTAAGCGAAACTGGCGGAACC, mL32R: TTGTTGCTCCCATAACCGATG, mcl-1F: AGAAAGCTGCATCCAACCAT, mcl-1R:CCAGCTCCTACTCCAGCAAC.

supplementary Figure legends Lindner et al.

Figure S1. Effect of S159 phosphorylation-deficient MCL-1 on neutrophil, monocyte, eosinophil and red blood cell numbers, and on cell proliferation

(A) Analysis of MCL-1 mRNA expression by quantitative RT-PCR in BM cells either before (left panel, freshly isolated BM or donor BM infected with empty vector pMIG, MCL-1 or MCL-1S159A as indicated) or six weeks after adoptive transfer (right panel, bars representing BM isolated from a representative mouse cohort expressing pMIG, MCL-1 or MCL-1S159Aas indicated). Bars represent relative MCL-1 mRNA expression, normalized to L32, error bars indicate SEM of biological replicates. (B) Neutrophil granulocyte numbers in the peripheral blood were determined using the ADVIA hematology analyzer 4 weeks after engraftment. Empty vector control pMIG 0.44 x 103/µl ± 0.04 (n= 29), MCL-1WT 0.72 x 103/µl ± 0.11 (n= 29) and MCL-1S159A 0.88 x 103/µl ± 0.14 (n= 30). (C) Monocyte count: pMIG 0.36 x 103/µl ± 0.12 (n= 29), MCL-1WT 0.35 x 103/µl ± 0.11 (n= 29) and MCL-1S159A 0.41 x 103/µl ± 0.1 (n= 30). (D) Platelets count: pMIG 581.8 x 103/µl ± 71.24 (n= 29), MCL-1WT 584.8 x 103/µl ± 57.77 (n= 29) and MCL-1S159A 636.3 x 103/µl ± 66.65 (n= 30). (E) Red blood cell count (RBC): pMIG 9.09 x 103/µl ± 0.1 (n= 29), MCL-1WT 9.55 x 103/µl ± 0.15 (n= 29) and MCL-1S159A 9.69 x 103/µl ± 0.13 (n= 30). (F) HeLa cell were stably infected with empty vector (blue), vectors encoding MCL-1WT-IRES-GFP (green) or Mcl-1S159A-IRES GFP (red) and sorted for GFP, indicating equal expression levels of MCL-1. Proliferation was monitored using the RTCA DP Analyzer. From the data of the cell growth curves, doubling times were calculated.

Figure S2: Cellular composition of pMIG, MCL-1WT and MCL-1S159A in BM, spleen, lymph nodes and thymus. Flow cytometric analysis of of primary and secondary hematopoietic organs. Total cells from mice of all three genotypes were isolated 6-8 weeks after engraftment. (A) Proportion of pro B (B220+IgM-CD43+), pre B (B220+IgM-CD43-), immature (B220+IgM+CD43-) and mature (B220hiCD43-IgM+) cells among GFP+B220+ BM cells (B) Proportion of splenic B (B220+) and T cells (CD3+) among GFP+ splenocytes (C) Proportion of LN B (B220+) and T cells (CD3+) among GFP+ LN lymphocytes (D) assessment of B cell maturation stages, bars represent percentage of transitional type 1 (T1) B cells (IgM+ IgD-), transitional type 2 (T2) B cells (IgM+ IgD+) and follicular (FO ) B cells (IgMlow IgD+) among GFP+B220+ splenocytes (E) CD4+ and CD8+ T cell subpopulations (DN (CD4-CD8-), DP (CD4+CD8+), CD4+CD8- and CD4-CD8+ SP maturation stages) among thymocytes (F) CD4+ and CD8+ T cell subpopulations among CD3+ splenocytes and (G) CD4+ and CD8+ T cell subpopulations among CD3+ LN lymphocytes. Mouse numbers (Fi. S2A-C, E) pMIG (n= 11), MCL-1WT (n= 9) and MCL-1S159A (n= 8); (Fig. S2D, F-I) pMIG (n= 26), MCL-1WT (n= 28) and MCL-1S159A (n= 28).(H) Splenocyte total cell extracts from mice, which had received BM expressing MCL-1 or MCL-1S159Awere analyzed by western blotting using anti-MCL-1 and anti-GFP antibodies.

Figure S3: Cooperation of S159 phosphorylation-deficient MCL-1 with Eµ-myc to induce lymphoma. (A) BM cells from young Eµ-myc transgenic mice were infected with pMIG, MCL-1WT or MCL-1S159A and analyzed by flow cytometry before engrafting into recipient mice. Peripheral white blood cell count was determined using the ADVIA hematology analyzer 4 weeks after engraftment. pMIG 7.37 x 103/µl ± 0.78 (n= 22), MCL-1WT 11.91 x 103/µl ± 1.44 (n= 22) and MCL-1S159A 15.23 x 103/µl ± 2.04 (n= 22). (B) Lymphocyte count: pMIG 4.59 x 103/µl ± 0.43 (n= 22), MCL-1WT 9.61 x 103/µl ± 1.39 (n= 22) and MCL-1S159A 13.08 x 103/µl ± 1.52 (n= 22). (C) Neutrophil granulocyte count: pMIG 1.22 x 103/µl ± 0.22 (n= 22), MCL-1WT 1.93 x 103/µl ± 0.51 (n= 22) and MCL-1S159A 2.33 x 103/µl ± 0.42 (n= 22). (D) RBC: pMIG 10.83 x 103/µl ± 0.23 (n= 22), MCL-1WT 10.95 x 103/µl ± 0.14 (n= 22) and MCL-1S159A 11.24 x 103/µl ± 0.23 (n= 22). (E) Platelets count: pMIG 772.4 x 103/µl ± 35.29 (n= 22), MCL-1WT 706.2 x 103/µl ± 46.52 (n= 22) and MCL-1S159A 751.8 x 103/µl ± 30.98 (n= 22). (F) Percentage of GFP+ peripheral blood cells, expressing MCL-1WT-IRES-GFP or MCL-1S159A-IRES-GFP as determined by flow cytometry.

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