Experimental mice
All experiments were conducted according to the guidelines of the Walter and Eliza Hall Institute of Medical Research Animal Ethics Committee or with the approval of the institutional animal care and use committee at St Jude Children's Research Hospital. Mcl-1+/- 26 and Puma-/- 6 mice were generated on a C57BL/6 background using C57BL/6-derived ES cells. Bim-/- 9, Bcl-x+/- 10
and a second strain of Mcl-1+/- mice 13 were generated on a mixed
C57BL/6x129SV background using 129/SV-derived ES cells and have been back-crossed to C57BL/6 mice for >10 generations. All gene-targeted animals carry the congenic marker Ly5.2 (CD45.2). Congenic C57BL/6-Ly5.1 (CD45.1) recipient mice were used for hematopoietic reconstitution experiments. Wild-type GFP-expressing (GFP+) competitor cells were derived from UBC-GFP transgenic mice 29. Rosa26-CreERT2 mice (referred to in the text and figures as ‘RosaCreER’ mice) on a C57BL/6 background have been described previously 30. Compound mutant mice were generated by intercrossing single knock-out mice to obtain animals of the desired genotypes.
Genotyping
The genotypes of all mice were determined by PCR using DNA extracted from tail clips taken at weaning (3 weeks). The tails were digested using 200 µL Direct Lysis tail buffer (Viagen Biotech) supplemented with proteinase K (Sigma-Aldrich) at 56°C for 6 h and heated to 85°C for 45 min to inactivate the proteinase K. One µL of the supernatant was added to 20 µL PCR reaction (GoTaq Green MasterMix, Promega) that was supplemented with 10 pmol of the appropriate oligonucleotide primers (sequences of primers will be provided on request).
Blood analysis
Blood was taken by retro-orbital bleed or at the time of sacrifice by cardiac puncture and analyzed using the Advia blood analyzer. Blood parameters were plotted using GraphPad Prism.
Soft tissues and sternum were harvested and fixed in 10% buffered Formalin and embedded in paraffin and stained with hematoxylin and eosin (H&E). Representative pictures of sternum were taken at a magnification of 400x (Figure 1) and 200x (Figure 2).
Supplementary Figure Legends
Supplementary Figure 1. Mcl-1+/-, Bcl-x+/- and wild-type mice were treated with one dose of 5-FU and white blood cell, platelet and reticulocyte counts in peripheral blood monitored over a 21-day period. n=15 (wild-type), 5 (Bcl-x+/-), 10 (Mcl-1+/-) mice; pooled results from 9 independent experiments. Data represent mean±SEM.
Supplementary Figure 2. Mcl-1+/-, Bcl-x+/- and wild-type mice were treated with one dose of 5-FU. B cell, T cell, monocyte and neutrophil numbers in the blood were monitored by flow cytometric analysis over a 21-day period. Antibodies against B220 and Thy-1 were used to identify B cells and T cells, respectively. Monocytes were identified as Mac1+Gr1lo; neutrophils were identified as Mac1+Gr1hi. n=15 (wild-type), 5 (Bcl-x+/-), 10 (Mcl-1+/-) mice; pooled results from 9 independent experiments. Data represent mean±SEM.
Supplementary Figure 3. The numbers of the indicated blood cell subsets were determined for untreated adult (12-16 weeks of age) wild-type and Mcl-1+/- mice.
Data represent mean±SEM, Mouse numbers (n) are indicated.
Supplementary Figure 4. (a) Bone marrow Lineage Marker-Sca-1+c-KIT+ (LSK) cells
from wild-type and Mcl-1+/- mice were analyzed using intracellular flow cytometry to determine their levels of MCL-1. Wild-type and Mcl-1-/- mouse embryonic fibroblasts (MEF) were used as positive and negative controls for the staining protocol, respectively. (b) Total bone marrow and LSK cell numbers were determined for
Mcl-1+/- and wild-type mice by flow cytometric analysis. (c) LSK cells were isolated from
Mcl-1+/-and wild-type mice by FACS sorting and treated in vitro with 4 Gy γ-irradiation or maintained in medium supplemented with IL-6, TPO, SCF and Flt3 alone. Cell survival was monitored over a 24 h period by flow cytometric analysis using staining with Annexin V and PI. Annexin V-PI- cells were deemed viable. n=3 mice for each genotype; data represent mean±SEM.
Supplementary Figure 5. Lethally-irradiated C57BL/6-Ly5.1 recipient mice were reconstituted with a 1-to-1 mixture of test Mcl-1+/-, Bcl-x+/- or wild-type bone marrow cells (all C57BL/6-Ly5.2) with wild-type (C57BL/6-Ly5.1) competitor bone marrow
cells (R1). An initial blood analysis was performed after 8 weeks. After 16 weeks a second blood analysis and analysis of hematopoietic tissues was performed. Moreover, bone marrow cells from three R1 reconstituted mice per genotype were pooled and used to reconstitute 3 lethally irradiated recipient (C57BL/6-Ly5.1) mice (reconstitution 2; R2). The test versus competitor cell derived contributions to the T cell pool and the splenic monocyte pool were determined by flow cytometric analysis for each round of reconstitution (R1 and R2) after 16 wks. Antibodies against Thy-1 and Mac-1 were used to identify T cells and monocytes, respectively. n=15 (wild-type), 4 (Bcl-x+/-, Mcl-1+/-) biological samples (each circle represents the mean of three replicate mice); pooled results from 7 independent experiments. Data represent mean±SEM.
Supplementary Figure 6. Lethally-irradiated C57BL/6-Ly5.1 recipient mice were reconstituted with a 1-to-1 mixture of Mcl-1+/- and wild-type (GFP+) bone marrow cells
The ratio within the LSK compartment was determined by flow cytometry initially prior to injection and then again after 15 h by harvesting bone marrow from the recipient mice and performing LSK staining. Each pair of samples is linked by a line, and data represent independent biological replicates (n=3).
Supplementary Figure 7. Test Bcl-x+/-, Bcl-x+/-;Bim+/-, Bcl-x+/-;Bim-/-, Bim+/-, Bim-/-,
Puma+/-, Puma-/- or wild-type (all C57BL/6-Ly5.2) bone marrow cells were mixed 1-to-1 with competitor wild-type (C57BL/6-Ly5.1-to-1) bone marrow cells and used to reconstitute lethally irradiated recipient (C57BL/6-Ly5.1) mice over two rounds of reconstitution (R1 and R2; R2 without addition of competitor bone marrow cells). The test versus competitor derived contributions within the bone marrow LSK population and within the B cell and monocyte populations in the blood of R1 and R2 recipient mice were determined at 16 weeks by flow cytometric analysis. The Bcl-x+/- and wild-type data are reproduced from Figure 3 to aid comparison to the other genowild-types. Antibodies against B220 and Mac-1 were used to identify B cells and monocytes, respectively. n=4 biological samples (each circle represents the mean of three replicate mice); pooled results from 13 independent experiments are shown. Data represent mean±SEM.
Supplementary Figure 8. Test Bcl-x+/-, Bcl-x+/-;Bim+/-, Bcl-x+/-;Bim-/-, Bim+/- Bim-/-,
Puma+/-, Puma-/- or wild-type (all C57BL/6-Ly5.2) bone marrow cells were mixed 1-to-1 with competitor wild-type (C57BL/6-Ly5.1-to-1) bone marrow cells and used to reconstitute lethally-irradiated recipient (C57BL/6-Ly5.1) mice over two rounds of reconstitution (R1 and R2; R2 without addition of competitor bone marrow cells). The test versus competitor-derived contributions to leukocytes in transplant recipients were determined by flow cytometric analysis in the T cell lineage (thymus, spleen), the B cell populations (bone marrow (BM) and spleen) and in the monocyte populations (bone marrow (BM) and spleen). The Bcl-x+/- and wild-type data are reproduced from Figure 3 to aid comparison to other genotypes. Antibodies against B220, Thy-1, and Mac-1 were used to identify B cells, T cells and monocytes, respectively. n=4 biological samples (each circle represents the mean of three replicate mice); pooled results from 13 independent experiments are shown. Data represent mean±SEM.
Supplementary Figure 9. Test Mcl-1+/-, Mcl-1+/-;Bim+/-, Mcl-1+/-;Bim-/-, Mcl-1
+/-;Puma+/-, Mcl-1+/-;Puma-/- or wild-type (C57BL/6-Ly5.2) bone marrow cells were mixed 1-to-1 with competitor wild-type (C57BL/6-Ly5.1) bone marrow cells and used to reconstitute lethally-irradiated recipient (C57BL/6-Ly5.1) mice over two rounds of reconstitution (R1 and R2; R2 without addition of competitor bone marrow cells). The test versus competitor-derived contributions to leukocytes were determined by flow cytometric analysis within the B cell, T cell and monocyte populations in the blood. The Mcl-1+/- and wild-type data are reproduced from Figure 3 to aid comparison to other genotypes. Antibodies against B220, Thy-1, and Mac-1 were used to identify B cells, T cells and monocytes, respectively. n=4 biological samples (each circle represents the mean of three replicate mice); pooled results from 9 independent experiments are shown. Data represent mean±SEM.
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