a) FACS-dependent separation of c-Myc-eGFP^Hi and c-Myc-eGFP^Lo LSK populations. b) CFU in vitro cultures using sorted c-Myc-eGFP^Hi and c-Myc-eGFP^Lo cells. In black: First plating of LSKs, in grey: second plating. Error bars indicate standard deviation (Std) (n=3 mice in 3 independent experiments). c) Peripheral blood chimerism in competitive reconstitution assays at 25 weeks post transplant (n=3 mice). d) Relative chimerism of sorted c-Myc-eGFP^Hi and c-Myc-eGFP^Lo LSKs (CD45.2) in the bone marrow (LSK subset), thymus and spleen (d) 17 weeks post transplantation (n=3 mice).

a) Heat map demonstrating relative gene expression signatures of c-Myc-eGFP^Hi and c-Myc-eGFP^Lo subsets. A direct comparison to the relative gene expression in Fbw7^−/− LSKs is also shown. b) Representative GSEA profiles showing positive correlation of the c-Myc-eGFP^Lo signature to stem cell gene sets and negative correlation to cell cycle and DNA replication gene sets. c) Representative GSEA profiles showing a positive correlation between both TGF-β and Wnt signaling pathways with c-Myc-eGFP^Lo gene expression data set.

a) FACS analysis showing detection of c-Myc-eGFP in different progenitor populations. LSK, MP and thymic DN (CD4⁻8⁻) subsets are shown. b) FACS analysis showing stabilization of c-Myc-eGFP protein in LT-HSC subset (LSK, CD150⁺, CD48⁻) upon Fbw7 deletion c-d) Deletion of a single Myc allele significantly rescues the Fbw7^−/− HSC phenotype. FACS profiles of bone marrow LSKs (c), cell cycle status of LSKs (d) and methylcellulose assays (CD150⁺ LSKs) (e) are shown. Black: first plating, Grey: second plating. Error bars indicate standard deviation (Std) (n=5 mice). Plots are a representation of at least 3 independent experiments.

a) GSEA analysis showing a positive correlation of c-Myc-eGFP^Hi gene expression profiles to a direct c-Myc target Chip-on-chip dataset. b) ChIP assay and Heat map of selected genes that are over-expressed in the c-Myc-eGFP^Hi cells. Red color: gene upregulation, Blue color: gene down-regulation. Two individual chromatin IPs using T-ALL cell line genomic DNA are shown. c) ChIP assay of selected genes using genomic DNA from purified Lineage⁻ c-Kit^eGFP+ bone marrow progenitor cells. Also, Heat Map showing over expression of selected genes in the c-Myc-eGFP^Hi LSK subset. Error bars indicate standard deviation (std) from 3 independent experiments.

a) Levels of c-Myc-eGFP expression during early hematopoiesis in Lineage⁺, Lineage⁻, LSK (Lineage⁻, c-Kit⁺, Sca1⁺) and MP (lineage⁻, c-Kit⁺, Sca1⁾ BM subsets. n=6 mice b) Immuno-fluorescence tracing of nuclear c-Myc-eGFP expression in Lineage⁻ bone marrow progenitors. c) Western blot demonstrating presence of c-Myc protein in c-Kit⁺ and absence in c-Kit⁻ subset of adult bone marrow. d) Overlay histogram depicting c-Myc-eGFP protein expression within LSK cells. e) c-Myc-eGFP expression in eGFP^−/−control, LT-HSC and MPP (1, 2) populations. f) qRT-PCR quantification of Myc and Fbw7 mRNA expression during HSC differentiation. g) Levels of c-Myc-eGFP protein correlate with the cell cycle status of LSK cells. Sorted c-Myc-eGFP^Hi or c-Myc-eGFP^Lo LSK cells were analyzed using Ki-67 and DAPI staining. Plots are a representation of at least 3 independent experiments.

a) mRNA transcript expression levels of Fbw7 (black), Myc (dark grey) and Nanog (light grey) in self-renewing (+LIF) and differentiating (−LIF, +RA) mouse ESC. Different days of differentiation (d0–6) are shown), b) Schematic diagram of Fbw7 gene-trap cassette. c) LacZ staining in Murine ESCs containing the Fbw7 gene trap cassette depicting upregulation of Fbw7 expression as Murine ESCs differentiate via the removal of LIF and the addition of Retinoic Acid (RA). d) Western blot showing stabilization of both phospho- (T58) and total-c-Myc in self-renewing and differentiating Murine ESCs treated with 20uM of proteosome inhibitor MG-132. e) ChIP assay was carried out using the specific regulators shown in Fig 6 revealing substantial enrichment upon c-Myc immunoprecipitation in Murine ESCs. Error bars indicate standard deviation (std) from 3 independent experiments.

a) FACS staining defining LSK cells in e.d.14.5 fetal liver and adult (6wk old) bone marrow. b) Cell cycle status of fetal and adult LSK cells. c) Levels of c-Myc-eGFP protein expression in fetal and adult LSK cells. The overlay histogram shows induction of c-Myc protein expression in fetal LSKs. d) Levels of c-Myc-eGFP protein expression in CD150⁺ LSKs. e) Methylcellulose culture using the indicated cell populations purified from the fetal liver (fetal) or the bone marrow (adult). Black: first plating, Grey: second plating. Error bars indicate standard deviation (Std) (n= 6 mice). f) c-Myc-eGFP^Lo but not c-Myc-eGFP^Hi expressing fetal liver LSK subsets show chimerism in the peripheral blood 20wks post transplant in competitive reconstitution assays. CD45.2⁺ cells are donor-derived cells in the peripheral blood (n= 6 mice). Plots are a representation of at least 3 independent experiments.

a) Inhibition of proteasomal degradation via MG-132 treatment resulted in accumulation of c-Myc protein in Myc^eGFP/− Murine ESCs. b) Visualization of c-Myc-eGFP in W4 and Myc^eGFP/− Murine ESCs with and without treatment with MG-132. GFP (green), nuclear staining (DAPI) c) Knock-down of Fbw7 using siRNA in c-Myc^eGFP/− ESCs resulted in accumulation of c-Myc protein (increase in eGFP levels) while siRNA targeting both Myc and GFP showed a reduction in c-Myc-eGFP levels as assessed by FACs analysis. d) qRT-PCR of Fbw7 mRNA expression in ESCs expressing shRNAs against Fbw7 and Nanog. A non silencing shRNA is used as a control. e) Western blot depicting accumulation of both phospho (T58)-and total-c-Myc protein upon shRNA mediated knock down of Fbw7 when compared to a non-silencing shRNA control. f) Alkaline phosphatase staining in Murine ESCs showed no difference in differentiation capacity upon shRNA mediated knock-down of Fbw7 when compared to non-silencing control while silencing of Nanog resulted in complete differentiation. g) siRNA knock-down of Fbw7 in a Nanog-eGFP reporter ESC line resulted in no difference in Nanog-eGFP expression levels when compared to non-silencing control. Knock-down of Oct4 resulted in a reduction of Nanog-eGFP expression levels (marking differentiation). Plots are a representation of at least 3 independent experiments