Fig. 5

The hypoxia-induced stemness of OC cells depends on PLD2 expression. (A) Heatmaps showing the z scores of the expression of stemness-associated genes obtained from TaqMan arrays. Genes are sorted according to decreasing z scores in Ev-carrying cells under normoxia. (B) Heatmaps showing the z-scores of stemness genes expression levels in SKOV3 cells carrying EV or plasmid overexpressing PLD2 under normoxia and carrying EV or plasmid expressing shPLD2 under hypoxia. Hierarchical clustering of the samples of (A) is shown. (C) Expression levels of SOX2, NANOG, CD44 and EPCAM stemness-associated genes in SKOV3, OVCAR8 and ES-2 cells carrying Ev and expressing PLD2, shPLD2 or both under normoxic or hypoxic conditions. The mRNA expression was calculated as 2^−∆Ct relative to the ACTB gene. (D) Quantification of the number and size of tumorspheres formed by SKOV3, OVCAR8 and ES-2 cells carrying Ev and expressing PLD2, shPLD2 or both. (E) Top, determination of the Sox9, Notch1, Sox2, and Sox17 protein levels by immunofluorescence in tumorspheres formed by OC cell lines carrying Ev and expressing PLD2, shPLD2 or both. Bottom, quantification of the percentage of cells with Sox9 and Notch1 expression in tumorspheres formed by ovarian cancer cell lines carrying Ev and expressing PLD2 or shPLD2. Scale bars: 100 μm. (F) Expression levels of SOX9, NOTCH1, SOX2 and SOX17 stemness-associated genes in tumorspheres formed by ovarian cancer cell lines carrying Ev and expressing PLD2, shPLD2 or both. The mRNA expression was calculated as 2^−∆Ct relative to the ACTB gene. A minimum of three biological were performed per each experiment. The data were analysed using Student’s t test. Asterisks indicate statistical significance with respect to Ev carrying cells, unless indicated by horizontal lines. *, P < 0.05; **, P < 0.01; ***, P < 0.001