Fig 2: Summary scheme of the identified paracrine signalling from breast cancer (BC) cells to macrophages. Briefly, breast cancer cells expressing high levels of inhibitor of differentiation 4 (ID4) protein produce vascular endothelial growth factor A (VEGFA) and other factors implicated in the induction of an angiogenic programme in neighbouring macrophages. In parallel to the induction of angiogenesis-related messenger RNAs, we observed a decrease of miR-15b/107 group members, with consequent release of the expression of its targets, as transcription factor hypoxia-inducible factor (HIF)-1A and granulin (GRN). TAM Tumour-associated macrophage

Fig 3: DAPK promotes HIF-1a degradation and DAPK deficiency increases HIF-1a levels in T cells.(a) HIF-1a deficiency impairs Th17 differentiation. WT and Hif1a-/- Th17 cells were re-stimulated and expression of IL-17A and IFN-? determined. (b) Enhanced HIF-1a protein levels in Dapk-/- Th17 cells. HIF-1a protein contents were determined in Th17 cells differentiated for 3 days (left). HIF-1a levels were quantified and normalized to tubulin (right). Mean±s.e.m., n=3. **P<0.01 for unpaired t-test. (c) DAPK-knockout does not affect HIF-1a mRNA induction during Th17 differentiation. The quantities of Hif1a transcript were determined in WT and Dapk-/- T cells in the course of Th17 differentiation. Mean±s.e.m., n=3. (d) DAPK induces HIF-1a protein downregulation in normal T cells. Activated primary T cells were transduced with DAPK-FLAG. GFP+ T cells were sorted, incubated under hypoxic conditions for 48 h, and the levels of DAPK-FLAG and HIF-la were determined. (e) DAPK reduces HIF-1a protein stability. HEK293T cells were transfected with DAPK and HIF-la, treated with CHX (50 ng ml-1) 48 h later, and the levels of HIF-1a and DAPK were measured. HIF-1a levels were quantified. (f) DAPK deficiency increases HIF-1a protein stability in T cells. WT and Dapk-/- CD4+ T cells were differentiated into Th17 cells for 2 days, followed by CHX (25 ng ml-1) treatment. The levels of HIF-1a protein were determined by immunoblots and were quantified. (g) DAPK-induced HIF-1a degradation is inhibited by MG132. HEK293T cells were transfected with DAPK and HIF-la. Forty-eight hours later, cells were treated with 10 µM MG132, 200 mM NH4Cl, 100 µg ml-1 leupeptin, or 100 µM chloroquine for 6 h. The HIF-1a levels were determined. (h) DAPK-induced HIF-1a degradation is proteasome-dependent. WT and Dapk-/- CD4+ T cells were differentiated into Th17 cells for 2 days, followed by MG132 (5 µM) treatment for 2 h. The levels of HIF-1a were determined. (i) DAPK deficiency increases the induction of HIF-1a protein in T cells. WT and Dapk-/- T cells were activated with CD3/CD28 under normoxic conditions, and the levels of HIF-1a at the indicated time points were determined. Data are representative of three (a,c,e–i) or two (d) independent experiments.

Fig 4: HIF-1a-knockout prevents excess Th17 differentiation in Dapk-/- T cells.Naive CD4+ T cells from WT, Dapk-/-, Hif1a-/- and Dapk-/-Hif1a-/- mice were allowed to differentiate into Th0 and Th17 cells. (a) The levels of DAPK and HIF-1a were determined in Th17 cells and Th0 cells after 3 days of differentiation. (b,c) HIF-1a deficiency reduces the excess Th17 differentiation in Dapk-/- T cells. TPA/A23187-stimulated production of IL-17A and IFN-? was examined in Th17 cells after 5 days of differentiation (b), and the contents of HIF-1a and ROR?t were determined (c). Data are representative of three (b) or two (a,c) independent experiments. (d) HIF-1a deficiency prevents exacerbated EAE generation in Dapk-/- mice. EAE was induced in WT, Dapk-/-, Hif1a-/- and Dapk-/-Hif1a-/- mice as in Fig. 1a. Mice were monitored for clinical signs of paralysis. Values are mean±s.e.m., *P<0.05, **P<0.01, ***P<0.001 for unpaired t-test.