Fig. 4

PLD2 promotes tumorigenesis and CSC-like features in ovarian cancer. (A) Analysis of the expression of PLD2 by RT‒qPCR in SKOV3, OVCAR8 and ES-2 cells carrying an empty vector (Ev) expressing PLD2 or shPLD2. The mRNA expression was calculated as 2^−∆Ct relative to the ACTB gene. (B) Western blot showing the protein levels of PLD2 in cells carrying Ev and expressing PLD2 or shPLD2. (C) Quantification of the number of colonies formed by SKOV3, OVCAR8 and ES-2 cells carrying Ev and expressing PLD2 or shPLD2. (D) Growth curve of SKOV3, OVCAR8 and ES-2 cells carrying Ev (light green) or expressing PLD2 (dark green) or shPLD2 (blue) represented as the accumulation of the doubling times. (E) Tumor growth in xenografts from SKOV3 and OVCAR8 cells carrying Ev, overexpressing PLD2 or shPLD2 were injected into female athymic nude mice. Cohorts of 5 mice each were used. (F) Percentage of paraclones, meroclones and holoclones formed by SKOV3, OVCAR8 and ES-2 cells carrying Ev expressing PLD2 or shPLD2. At least 200 individual clones were analysed. The averages and SDs of three independent experiments are shown. (G) Top, Representative images of tumorspheres formed by SKOV3, OVCAR8 and ES-2 cells carrying Ev expressing PLD2 or shPLD2. Bottom, quantification of the number and size of tumorspheres. Scale bars: 250 μm. (H) Analysis of the expression by RT‒qPCR of stemness-associated genes and PLD2 in total cell extracts and tumorspheres from SKOV3, OVCAR8 and ES-2 cells carrying Ev and expressing PLD2 or shPLD2. The mRNA expression was calculated as 2^−∆Ct relative to the ACTB gene. The average and SD of at least three independent experiments are shown. The data were analysed using Student’s t test. Asterisks indicate statistical significance with respect to Ev carrying cells. *, P < 0.05; **, P < 0.01; ***, P < 0.001