Fig. 6: Lactate directly binds and inhibits the activity of PHD2.

a Egln1 mRNA level in macrophages with 20 mM lactate or NaCl. n = 4 biologically independent samples. b mRNA of Egln1 in epididymal white adipose tissue (eWAT) of wildtype (WT) and adipocyte-specific Ldha knockout (AKO) mice. STC-WT: n = 4; STC-AKO: n = 4; HFD-WT: n = 8; HFD-AKO: n = 8 biologically independent animals. c In vitro fluorometric PHD2 activity assay in the presence of 20 mM lactate or NaCl. n = 4 biologically independent samples. d His-tagged HIF-1α was incubated with PHD2 in presence of 20 mM lactate or NaCl. Time-dependent HIF-1α hydroxylation was examined by Western blotting. e Pull down assay between PHD2 and lactate. His-tagged PHD2 protein was incubated with ¹⁴C-labeled lactate. Unlabeled lactate or α-ketoglutarate (α-KG) was added where indicated. Count per min (CPM) in Ni-NTA beads was measured by liquid scintillation counter. Group 1: n = 3; Group 2,3,4,5: n = 4 biologically independent samples. f Isothermal titration calorimetry (ITC) of PHD2 with lactate and α-KG. Sequential heat pulses for each injection (upper panel) and the integrated data (lower panels). α-KG → PHD2: injecting α-KG to PHD2; Lac→PHD2: injecting lactate to PHD2; α-KG → PHD2 + Lactate: injecting α-KG to PHD2 + lactate mixture. g, h Representative images of autodocking for catalytic domain of PHD2, lactate and α-KG. Blue: α-KG, Green: lactate, yellow dash lines: hydrogen bonds. i, j Egln1 gene was knocked out by Crispr/Cas9 mediated method in immortalized mouse BMDM. The macrophages were treated with lactate or vehicle control. i Western blotting of PHD2 in WT and Egln1 KO macrophage. j mRNA level of Il-1β in WT and Egln1 KO macrophages with 20 mM lactate or NaCl. n = 4 biologically independent samples. Data represent mean ± SEM; Significance was calculated by two-way ANOVA with post hoc Bonferroni correction (a, b, e, j) or two-tailed student’s t test (c).