P4HA1 requires HIF1α for its transcriptional activation, which could be disrupted by ATF3 overexpression (OE). (A) A volcano plot showing differentially expressed genes with statistical significance and fold-change (FC) between the normoxia and hypoxia groups. Significantly affected genes were selected by their FCs (≥2 or≤–2) and adjusted P-value (<.05). Each dot denotes a gene, and P4HA1 is remarked. (B) A heatmap shows the significant upregulation of genes in A. (C) qRT-PCR result of P4HA1 mRNA level under the normoxia and hypoxia groups. (D and E) ChIP analysis for the binding of HIF1α to the P4HA1 gene promoter in U87MG (D), and TBD0220 (E) cells. (F) Co-IP assays were conducted with anti-p300 antibody under normoxia or hypoxia conditions. (G) Co-IP assays were conducted with anti-p300 antibody with or without ATF3 overexpression under hypoxia conditions. (H) qRT-PCR result of P4HA1 mRNA level with or without ATF3 OE under hypoxia condition. (I and J) ChIP analysis of the binding of HIF1α to the promoter region of P4HA1 gene in U87MG (I) and TBD0220 (J) cells under hypoxic conditions with or without ATF3 overexpression. (K) Illustration of the potential HIF1α/ATF3-mediated regulation of P4HA1. Error bars represent mean ± SEM; n = 3 independent experiments. P-values are based on two-way ANOVA. ^*P < .05, ^**P < .01, ^***P < .001, ^****P < .0001 and n.s. indicated no significant difference.