Fig. 3: PADI4-mediated citrullination of HIF-1α promotes HIF-1α stability and transactivation.

a Hep3B (left panel) and HepG2 cells (right panel) expressing Flag-EV or Flag-PADI4 were cultured under normoxic or hypoxic conditions for 6 h, followed by western blotting analysis. The samples were derived from the same experiment, but different gels for HIF-1α, Flag, Actin, and another for PADI4 were processed in parallel. b Hep3B and HepG2 cells expressing NTC or PADI4 shRNAs were cultured under normoxic or hypoxic conditions for 6 h, followed by western blotting analysis. c Hep3B cells were treated with DMSO or 1 or 2 μM BBCA for 24 h, followed by hypoxic treatment for another 6 h before western blotting analysis. d Hep3B cells were treated with DMSO or 2 μM BBCA for 24 h, followed by hypoxic treatment for another 24 h. The mRNA levels of LDHA and PDK1 were analyzed by qPCR. e Western blot analysis of HIF-1α and PADI4 protein levels in HepG2 cells expressing NTC and PADI4 shRNAs cultured under normoxic or hypoxic conditions in the presence or absence of 10 μM MG132 for 6 h. f Western blot analysis (left panel) of HIF-1α protein levels in HEK293T (upper panel) and Hep3B cells (lower panel) expressing Flag-HIF-1α^WT or Flag-HIF-1α^R698A treated with 20 μg/ml CHX for the indicated times. Quantification of HIF-1α protein levels relative to Actin. g, h Endogenous PADI4-knockdown Hep3B cells were infected with viruses expressing Flag-EV, Flag-PADI4^WT, Flag-PADI4^D473A, or Flag-PADI4^C645A and further cultured under normoxic or hypoxic conditions for 6 h. Cell lysates were harvested, and the protein and mRNA levels of HIF-1α were analyzed by western blotting (g) or qPCR (h), respectively. i The cell lines described in Fig. 3g were cultured under normoxic or hypoxic conditions for 24 h. The mRNA levels of LDHA, PDK1, and PGK1 were analyzed by qPCR. Immunoblots are representative of three independent experiments (a–c, e–g). Data were presented as mean ± SEM of three independent experiments (d, f, h, i). Statistical analyses were performed by ordinary one-way ANOVA (d, h, i) or two-way ANOVA test (f) with Turkey’s multiple comparisons test. Source data are provided as a Source Data file.