Table S1. Reagents used in this work. Table S2: Patient Cohort characteristics. Figure S1. PLD2 expression in OC patients and patient survival in OC public databases. (A) PLD2 expression in the OC patient databases GSE12172, GSE9891, GSE63885 and GSE2109 indicating OC subtype. (B) PLD2 expression in the OC patient databases GSE18520, GSE40595, and GSE38666, indicating OC subtype. (C) Kaplan-Meier plots showing overall survival (OS) of patients with high (red) or low (green) PLD2 expression levels in three OC databases with survival data: GSE13876 (advanced HGSOC); GSE23554 (advanced serous epithelial OC); and GSE31245 (92% serous, 2% endometroid, 6% clear cell). Data were analyzed with the log-rank test, and the associated P-values are shown in the graphs. (D) Kaplan-Meier plots showing overall survival (OS) of patients with high (red) or low (green) risk. PLD2 expression for each group is shown on the right of each graph. (E) Kaplan-Meier plots generated with Kaplan-Meier Plotter showing PFS (left column), PPS (middle column) and OS (right column) by splitting patients according to PLD2 expression in all OC patients (top row) or only HGSOC patients (bottom row). Expression levels are shown as log2 transformed values from the R2 database. Data were analyzed using Student’s t-test. *, P <0.05; **, P < 0.01; ***, P < 0.001. Figure S2. HIF1a levels in OC cell lines in response to hypoxia. (A) Representative images of HIF-1a protein levels by immunofluorescence in SKOV3, OVCAR8 and ES-2 cells under normoxia and hypoxia or in the presence of the HIF-hydroxylase inhibitor DMOG. (B) Western blot showing HIF-1a and alpha-tubulin protein levels in SKOV3, OVCAR8 and ES-2 cells under normoxia and hypoxia or in the presence of the HIF-hydroxylase inhibitor DMOG. A minimum of three independent experiments were performed. Figure S3. Differential analyses of chromatin accessibility in OC cells. (A) Volcano plot showing differential analyses of chromatin accessibility between SKOV3 cells carrying Ev and plasmid overexpressing PLD2 in normoxia. (B) Heatmaps and average profiles plotting normalized ATAC-seq signal in SKOV3 cells carrying Ev and overexpressing PLD2 for the differentially accessible regions (DARs) in (A). (C) Gene Ontology term enrichment analyses of biological processes for the genes associated with DARs in SKOV3 cells carrying EV in normoxia versus hypoxia. (D) Gene Ontology term enrichment analyses of biological processes for the genes associated with DARs in SKOV3 cells carrying Ev versus overexpressing PLD2 in normoxia. Figure S4. Motif and footprint analyses of transcription factor binding in OC cells. (A-B) Motif enrichment analyses of the increased and decreased ATAC peaks in OC cells carrying Ev in hypoxia vs. normoxia (A) and +/- PLD2 expression in normoxia (B). The three motifs with the lowest p values are shown in each case. (C) Venn diagrams plotting the overlap between TFs with increased binding in hypoxia and expressing PLD2 in normoxia (top) or the overlap between TFs with increased binding in hypoxia and expressing shPLD2 in hypoxia (bottom). (D) Distribution of fold changes in the ATAC peaks containing the motifs FOS::JUN in Ev normoxia vs. Ev hypoxia (top) or in Ev hypoxia vs. shPLD2 hypoxia. (E) Aggregate footprint signal of the peaks containing the motifs in (D). Figure S5. Clustering of differentially accessible regions in OC cells. (A) Heatmaps plotting normalized ATAC-seq signal at peaks associated with stemness genes in SKOV3 cells carrying Ev or plasmid expressing PLD2 in normoxia and carrying Ev or plasmid expressing shPLD2 in hypoxia, for the differentially accessible regions (DARs) clustered using k-means method in 4 clusters. (B) Tracks with ATAC-seq in SKOV3 cells carrying Ev or expressing PLD2 in normoxia and carrying Ev or shPLD2 in hypoxia, at the JAG1 locus. Figure S6. Expression of stemness and hypoxia genes correlated with PLD2 in OC patients. Heatmaps showing the expression z-scores of stemness-associated genes or hypoxia-response genes whose expression correlated with PLD2 in GSE40595 and GSE38666 OC patient databases. Figure S7. Hypoxia induces CSCs in ovarian cancer cells. (A) Top, Representative images of tumorspheres formed by SKOV3, OVCAR8 and ES-2 cells in normoxia or hypoxia. Bottom, quantification of the number and size of tumorspheres. Scale bars: 250 μm. (B) Percentage of holoclones formed by SKOV3, OVCAR8 and ES-2 cells in normoxia or hypoxia. At least 200 individual clones were analyzed. (C) Analysis of the expression of NANOG, SOX2, CD44 and EPCAM stemness-associated genes by RT-qPCR in SKOV3, OVCAR8 and ES-2 cells in normoxia or hypoxia. The mRNA expression was calculated as 2-∆Ct relative to the ACTB gene. (D) Percentage of CD133 positive cells measured by FACS in SKOV3, OVCAR8 and ES-2 cells in normoxia and hypoxia The average and SD of three independent experiments are shown in all cases. A minimum of three independent experiments were performed and the data were compared using Student’s t tests. Asterisks indicate statistical significance with respect to normoxia. *p < 0.05; **p < 0.01; ***p < 0.001. 13 Figure S8. Relative protein quantification of PLD2 normalized to alpha-tubulin from the western blots in Figure 4B. Figure S9. PLD2 expression and clone analyses. (A) Western blot showing PLD2 and alpha-tubulin protein levels in SKOV3, OVCAR8 and ES-2 OC cells carrying Ev, a plasmid expressing shPLD2 or plasmids expressing shPLD2 and PLD2. (B) Percentage of paraclones, meroclones and holoclones formed by SKOV3, OVCAR8 and ES-2 cells carrying Ev or plasmids expressing PLD2, shPLD2 or both in hypoxia or normoxia. At least 200 individual clones were analyzed. The average of three independent experiments is shown. A dotted line represents the percentage of holoclones in Ev carrying cells as a reference. Data were compared using Student’s t tests. Asterisks indicate statistical significance with respect to Ev carrying cells. *p< 0.05. (C) Left, determination of PLD2 protein levels by immunofluorescence in tumorspheres formed by OC cells carrying Ev and expressing PLD2 or shPLD2. Right, quantification of the percentage of cells with PLD2 expression in tumorspheres. Scale bars: 100 μm. Figure S10. Analyses of the EMT in OC cells in response to hypoxia and/or PLD2 expression. (A) Heatmaps showing the expression z-scores of epithelial-to-mesenchymal transition-associated genes obtained from TaqMan Arrays. Genes are sorted according to decreasing z-scores in the Ev-carrying cells under normoxia. (B) Heatmaps showing the z-scores of EMT genes expression levels in SKOV3 cells carrying EV or plasmid overexpressing PLD2 under normoxia conditions and carrying EV or plasmid expressing shPLD2 under hypoxia condition. Hierarchical clustering of the samples is shown. (C) Expression levels of SNAI1, VIM, CDH1 and CDH2 EMT-associated genes in cells carrying Ev or plasmids expressing PLD2 under normoxia or shPLD2 under hypoxia conditions. (D) Left, representative images of the Boyden chamber migration assays in SKOV3 and OVCAR8 cells carrying Ev or plasmids expressing PLD2 or shPLD2 under normoxic or hypoxic conditions. Right, quantification of the Boyden chamber migration assays. A minimum of three independent experiments were performed and the data were analyzed using Student’s t-test. *, P < 0.05; **, P < 0.01; ***, P < 0.001.

Supplementary Material 1: Essential role of PLD2 in hypoxia-induced stemness and therapy resistance in ovarian tumors.

Funding: Consejo Superior de Investigaciones Cientificas (CSIC).

Peer reviewed