Fig. 4.

P4HA1 enhances HIF-1α stability by modulating α-KG l and succinate levels. a Scheme showing that HIF-1α hydroxylation is regulated by oxygen tension and metabolites (α-KG and succinate). b–d Cytoplasmic α-KG levels were measured in control, P4HA1-silenced MDA-MB-231, and P4HA1-expressing cells. Results are presented as mean ± SEM; n = 3; *p < 0.05, **p < 0.01, independent Student’s t test. e Cytoplasmic succinate levels were measured in control and P4HA1-silenced MDA-MB-231 cells. Results are presented as mean ± SEM; n = 3; **p < 0.01, independent Student’s t test. f, g HIF-1α protein levels were assessed in control, P4HA1-expressing MCF10A, and P4HA1-silenced cells in the presence or absence of octyl-α-ketoglutarate (1 mM) or dimethyl-succinate (4 mM). h, i ODD-luciferase reporter activity was measured in control, P4HA1-silenced, and P4HA1-expressing cells in the presence or absence of octyl-α-ketoglutarate (1 mM) or dimethyl-succinate (4 mM); n = 3, results are presented as mean ± SEM. *n = 3; p < 0.05, one-way ANOVA test. j HIF-1α protein levels were assessed in control, wild-type P4HA1-expressing MCF10A cells, and hydroxylation-deficient P4HA1 mutant-expressing MCF10A cells