Imtiyaz et al., Fig. S1

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1 . Imtiyaz et al., Fig. S % O.1.5 Lin/Sca-1/IL-7Rα GMPs.17 MPs.3 Days 3% O.1 MEPs.35 D3 Days 1, N, N, H, H 1 1 Days Supplemental Figure S1. Macrophage maturation, proliferation and survival are not dependent on HIF-α. () Myeloid progenitors are not influenced by HIF-α. The IL-7Rα - Lin - Sca-1 - c-kit + fraction (gated, top panel) was subdivided into MPs (FγRII/III lo D3 + ), GMPs (FγRII/III hi D3 + ), and MEPs (FγRII/III lo/- D3 - ) based on their D3 and FγRII/III expression. Representative panels of FS analyses are shown. () Normal proliferation of Hif- α MDMs. MDMs treated with normoxia (1% O ) and hypoxia (3.% O ) and their proliferation was determined by counting cell numbers every two days for eight days. () Normal survival of Hif-α MDMs. MDMs were placed under normoxia (1% O ) and hypoxia (.5% O ) and their viability was analyzed by propidium iodide uptake assay.

2 Imtiyaz et al., Fig. S TP : TP : D TP : E N TP H H+TP F M Spleen P D11b Supplemental Figure S. Neuthophils do not express HIF-α. () Expression of HIF-1α and HIF-α in bone marrowderived mouse neutrophils was evaluated at normoxia and relative mrn levels were normalized to that of endogenous HPRT1. () HIF-α protein stabilization in bone marrow-derived mouse neutrophils was compared to that in MDMs exposed to hypoxia for 3hrs. n.s.: non-specific band as a loading control. () MPRO cells were differentiated for 3 days in the presence of R (1 µm). HIF-α and HIF-1α mrn level was determined by QRT-PR following the stimulation with TP (1 ) for 15 min. (D) HIF-α protein level in MPRO cells was evaluated by Western blotting following TP treatment. (E) Surface expression of D11b on MPRO cells was analyzed by flow cytometry following TP treatment. (F) Normal development of neutrophils in Hifa mice. Neutrophils in the bone marrow, spleen and peripheral blood were quantified by immunostaining for D11b in combination with Gr.1. N: normoxia (1% O ); H: hypoxia (.5% O ); Macs: macrophages.

3 Imtiyaz et al., Fig. S3 loxp/1loxp, LysM-re loxp 1loxP (Δ) LPS : LPS : Macs Ds Ds Ds Ds LPS : Supplemental Figure S3. HIF-α is still expressed in Hifa dendritic cells. () Insufficient deletion of the Hif-α floxed (i.e. loxp) allele. DN was isolated from differentiated bone marrow-derived dendritic cells and subjected to PR analysis. D1 and D indicate primary dendritic cells generated from two independent mice with Hifa genotype, Tail DN obtained from Hifa mice was used as a control. () Dendritic cells were treated with LPS (1 ) for hrs under normoxia and hypoxia, and mrns of HIF-α and HIF-1α was quantified by QRT-PR. () HIF-α protein level in dendritic cells was evaluated by Western blotting following LPS treatment. Macs: macrophages; Ds: dendritic cells.

4 Imtiyaz et al., Fig. S , n.t., n.t., IL-, IL-, n.t., n.t., IL-, IL Supplemental Figure S. M responses of Hifa macrophages. () MDMs were treated with recombinant IL- for 3 hrs a concentrations indicated. rginase activity was measured using an isonitrosopropiophenone (ISPF)-based method. () Mannose receptor expression was determined by flow cytometry following hrs of IL- stimulation. () FN1 expression was evaluated by QRT-PR following hrs of IL- stimulation. IL- concentration used for () and () is 5. N: normoxia; H: hypoxia.

5 Imtiyaz et al., Fig. S5 1 1% O +/+ LPS: INF-γ: LPS: INF-γ:.5% O /+ Supplemental Figure S5. IL- production of M1-stimulated Hifa +/+ and Hifa MDMs under normoxia () and hypoxia () was determined by ELIS.

6 Supplemental Table S1: haracterization of H tumors in HIF-α LysM-re mice n value p value (, ) (t-test) Tumor number* 3. ± ±. (7, ). Tumor area (% tumor/liver area) 1. ± 5.5. ± 5.3 (1, 1).91 lood vessel number** Luminized 1.7E-5 ± 3.E- 1.9E-5 ± 3.1E- (15, 15).59 Non-luminized.9E- ± 7.1E-7 5.E- ±.E-7 (15, 15).9 * Total tumor number per liver section ** lood vessel number per unit tumor area