Figure 1.

Citrate treatment affects PC3 cell proliferation, survival, and metabolism. (a) Citrate impairs the adhesion of PC3 cells in dose-dependent manner. (b) Citrate inhibits the proliferation in PC3 cells: 5 and 10 mM citrate was added to a culture medium of PC3 cells at the time of seeding, and cells were counted after 48 h in a Neubauer hemocytometer. Data are expressed as mean ± S.D. of a representative experiment performed in triplicate. The differences between treated and untreated cells are statistically significant (p < 0.005 Anova followed by Bonferroni post-test). (c) PC3 cells were seeded, and citrate was added 24 h after plating. Cell number was determined after 72 h. Data represent the mean of quadruplicate values of two independent dishes. (d) Growth curves of PC3 and PC3 Cit20 after citrate withdrawal. Cell number was determined at the indicated time points. Data represent the mean of quadruplicate values of two independent dishes (p < 0.001 Anova followed by Bonferroni post-test). (e) Altered bioenergetic profile in citrate-resistant PC3 cells. Kinetic profile of ECAR in PC3 cells and PC3 cit20 (citrate-resistant) cells. Data are expressed as mean ± S.E.M. of three independent experiments, each of them in triplicate. ECAR was measured in real time, under basal conditions and in response to glucose, oligomycin, and 2-DG. (f–h) Lysates from PC3, PC3 Cit20, and PC3 Cit20 WD cells were analyzed by Western blot using the indicated antibodies. γ tubulin was used as loading control. * p ≤ 0.05; *** p ≤ 0.001, Student t-test.