Fig. 1

Hypoxia leads to the overexpression of PLD2 through HIF-1α in OC. (A-B) PLD2 expression in OC patient databases. Box plots show PLD2 expression as log₂ transformed values from R2 database in ovarian tumor or non-tumor (NT) tissue. (C) Kaplan–Meier plot showing overall survival (OS) of patients with high or low PLD2 expression in the database GSE19161. (D) PLD2, LDHA and VEGFA expression by RT-qPCR in SKOV3, OVCAR8 and ES-2 ovarian cancer cell lines under normoxia, hypoxia or treated with the HIF hydroxylase inhibitor DMOG. The mRNA expression was calculated as 2^−∆Ct relative to the ACTB gene. (E) Immunofluorescence and quantification of PLD2 protein levels in SKOV3, OVCAR8 and ES-2 cells carrying empty vector (Ev) under normoxia, hypoxia or treated with DMOG. Scale bars: 10 μm. (F) Top, heatmaps showing Micro-C signal in hESCs in a 0.5-Mb region of chromosome 17, TAD boundaries, the TAD containing PLD2 gene and ENSEMBL genes. Bottom, zoom within the TAD containing the PLD2 gene showing ENCODE cis-regulatory elements (CREs) and those containing HIF1 motifs with relative scores higher than 0.9. (G) Luciferase activity assay of PLD2 promoter and putative enhancer in HEK293 cells under hypoxia or normoxia. (H) Western blot showing PLD2, HIF-1α and alpha-tubulin levels in SKOV3 or OVCAR8 cells with or without a small interfering RNA (siRNA) of HIF-1α. (I) Western blot showing PLD2, HIF-1α, and alpha-tubulin protein levels in SKOV3 and OVCAR8 cells expressing a hif1a mutant (HIF mut) or Ev. (J) Immunofluorescence and quantification of PLD2 protein levels in SKOV3, OVCAR8 and ES-2 cells carrying Ev or expressing Hif Mut under normoxia or hypoxia. Scale bars: 10 μm. At least three biological replicates were performed per experiment. Data were compared using Student’s t tests. For D, E and G, asterisks indicate statistical significance compared with normoxia, and for J, compared with normoxia of cells carrying Ev. *p < 0.05; **p < 0.01; ***p < 0.001