(A)Smo^Null or Smo^WT mice (n = 2–3 per group) were treated with InvivoGen pIpC 200 μg and sacrificed 4 weeks after injection for complete endpoint analysis. Multiparameter flow cytometry analysis was used to identify myeloid progenitor (Prog) and LSK populations (top row) and the myeloid subsets CMP, GMP, and MEP (bottom row). There was no statistically significant difference between Smo^Null or Smo^WT groups in percentage of LSK cells (p = 0.5622) or myeloid progenitors (p = 0.2948) among live cells. Similarly, there were no differences in the percentage of CMP (p = 0.39), GMP (p = 0.5183), or MEP (p = 0.4823).
(B) Bar graph representation of data. Results were analyzed in an unpaired t test. Errors bars represent SEM.
(C) Fraction of LSK (left panel) or myeloid progenitors (right panel) in G0/G1 versus S/G2/M in overlapped cell-cycle histograms for SmoNull (blue) compared to SmoWT (red) mice.
(D) Cell-cycle analysis with Pyronin Y and Hoescht staining for LSK (left) and myeloid progenitors (right) shown as a percentage of live cells from the same animals as described in (A). An unpaired t test was used for analysis. Error bars represent SEM.

Flow cytometric analysis of whole bone marrow cells using CD45.1 and CD45.2 surface marker labeling to identify CD45.1-positive recipient (left upper quadrant), CD45.1/CD45.2 double-positive competitor (right upper quadrant), or CD45.2 donor (right lower quadrant) cell populations. Smo^WT/CD45.2 or Smo^Null/CD45.2 donor bone marrow was transplanted together with a competitor WT bone marrow from B6.SJL F1^{CD45.1/CD45.2} mice into B6.SJL^CD45.1 recipients (n = 15 per group). The following donor:competitor ratios were used: 1:0, 3:1, 1:1, 1:3, and 0:1 (n = 3 per subgroup). Recipient mice were treated with pIpC 3 weeks after engraftment. Donor-to-competitor chimerism analysis of the bone marrow at 16 weeks is shown for Smo^Null/CD45.2 (top) or Smo^WT/CD45.2 mice at representative donor:competitor ratios of 1:0 (left), 1:1 (middle), and 0:1 (right). Analysis of the peripheral blood and spleen showed similar results.

(A) Modulation of the Hedgehog signaling does not impact colony formation. Normal murine bone marrow cells (1.3 × 10⁵) per condition were plated in methylcellulose supplemented with SCF, IL-3, IL-6, and erythropoietin in the presence or absence of rSHH and/or the hedgehog antagonist HhAntag. There was no significant difference in CFU-GM, BFU-E, or CFU-GEMM when comparing vehicle versus HhAntag versus rShh agonist versus HhAntag + rShh (n = 3 per group, p = 0.5741, 0.6338, and 0.6935, respectively; one-way ANOVA test). Error bars represent SEM.
(B) Smo^Null or Smo^WT bone marrow cells were retrovirally transduced with MSCV-MLL-AF9/GFP and then serially replated on methylcellulose media for five rounds. Over the course of five rounds of replating, there was no significant difference in the absolute number of colonies observed (n = 7, p = 0.2424 using an unpaired t test). Error bars represent SD.
(C) Smo^Null or Smo^WT bone marrow cells retrovirally transduced with MLL-AF9/GFP were transplanted into lethally irradiated B6.SJL WT recipient mice. No significant difference in the disease latency or overall survival of Smo^Null compared to Smo^WT mice was noted (n = 5, p = 0.1448, log rank test). Results are shown in a Kaplan-Meier survival curve.
(D) Flow cytometric analysis demonstrating no difference in immunophenotype of Mac-1 and c-kit double-positive AML cells in Smo^Null compared to Smo^WT samples.

Bone marrow of Smo^Null or Smo^WT mice was harvested and transplanted into lethally irradiated B6.SJL recipients (n = 6 per group), which were treated with 25 μg/gram pIpC 3 weeks after engraftment. Thymi were harvested 12 weeks after pIpC treatment to analyze the T cell subpopulations and thymocyte development.
(A) Flow cytometry analysis was performed to identify CD4+ T helper cells, CD8 + cytotoxic T cells, and CD4+/CD8+ double-positive (DP) T cells, as well as thymocyte development (DN1-DN4) characterized by CD44 and CD25 characteristics. There was no difference between Smo^Null or Smo^WT mice in regards to T cell subpopulations (p = 0.9983) or thymocyte developmental stages (p = 1.0).
(B) Representative populations shown in flow scattergrams.
(C) Percentage of TCRαβ-rearranged cells gated on CD4+, CD8+, or CD4+/CD8+ T cells. There was no statistically significant difference between thymocytes obtained from Smo^Null or Smo^WT mice (p = 0.9917).
(D) Representative data for CD4+ and CD4+/CD8+ populations are shown in a histogram. An unpaired t test was used for statistical analysis. Error bars represent SEM.

Peripheral blood counts of Smo^Null or Smo^WT mice that received sequential 5-FU treatment 3 weeks after pIpC treatment. No differences in (A) WBC (n = 5, p = 0.7130), (B) Hematocrit (Hct) (n = 5, p = 0.9524), or (C) platelet count (n = 5, p = 0.6867) was observed over time. An unpaired t test was used for statistical analysis. Error bars represent SEM. (D) Survival outcome after sequential 5-FU treatment. 5-FU was administered i.p. weekly at a dose of 150 mg/kg in a survival study. Results were analyzed using a log rank test and expressed as Kaplan-Meier survival curves (n = 5, p = 0.7569).