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. Author manuscript; available in PMC: 2015 Mar 25.
Published in final edited form as: Nature. 2014 Jul 13;513(7519):559–563. doi: 10.1038/nature13490

Figure 3. Lactic acid is sufficient to induce Vegf and Arg1 via HIF1α.

Figure 3

a, b, Control (DMEM) or LLC-tumour-conditioned medium was used unfractionated (whole) or as <3-kDa or >3-kDa fractions to stimulate cells as follows. A luciferase reporter assay of 293T cells transfected with HIF1α oxygen-dependent domain (ODD)-luciferase was carried out to measure protein stabilization of the ODD; deferoxamine (DFO) was used as a hypoxia mimetic (a). Expression analysis by qPCR of Arg1 mRNA in bone-marrow-derived macrophages (b). c, Lactic acid concentration in the tumour-conditioned media from five tumour cell lines, collected after culturing at confluence for 4 days. d, Expression analysis by qPCR of Vegf mRNA in bone-marrow-derived macrophages stimulated with the tumour-conditioned media in c. e, f, Expression analysis by qPCR of Vegf (e) and Arg1 (f) mRNA in bone-marrow-derived macrophages cultured with a concentration gradient of lactic acid (LA). g, h, Expression analysis by qPCR of Vegf and Arg1 mRNA in wild-type (WT) and Hif1a−/− bone-marrow-derived macrophages. b–h, The histogram bars represent the expression level of three biological replicates (relative to expression in DMEM), displayed as mean ± s.e.m. *P < 0.0001; **P < 0.001, using a two-tailed, unpaired t-test. All experiments were performed at least twice. NS, not significant; RLA, relative luciferase activity.