Fig. 4: Differential secretion of spermidine synthase contributes to the non-cell autonomous anti-proliferative effect of PGC1α in prostate cancer.
A Incorporation of 13C from U-13C5-L-Methionine (2 h pulse) into spermidine and spermine metabolites after 3 day-treatment of PC3 cells grown with the indicated CM. B Validation of SRM overexpression in PC3 cells with inducible expression of PGC1α (Western blot, one representative image out of 4). C Quantification of 2D-cell proliferation (crystal violet) of PC3 grown with differential CM produced by: PGC1α non-expressing and expressing PC3 cells with or without overexpression of SRM (n = 4). D Quantification of PC3-luc cells co-injected in nude mice together with PGC1α non-expressing (No Dox) and expressing (Dox) PC3 cells with or without overexpression of SRM, at day 6 (left panel) and 16 (right panel). (n = 10 tumors per group; 2 injections per mice). E Confirmation of SRM silencing in PC3 cells using two independent short hairpin RNAs (Western blot, one representative image out of 3). F Quantification of 2D-cell proliferation (crystal violet) of PC3 grown with differential CM produced by PC3 cells in which the expression of SRM was silenced (n = 3). In C, E, a representative image of the crystal violet staining is included beside and below the quantification, respectively. In C, F, data are normalized to the CM -Dox (non-PGC1α expressing) condition (C) or to the CM shScr (F), depicted by a black dotted line. P producer cells, R recipient cells, CM conditioned media, D or Dox doxycycline, FC fold change. n.s not significant. Statistics: one sample t-test with reference value 1 (C, F), unpaired-t-test (A, C), Mann Whitney test (D). Asterisks and dollar symbols indicate statistical differences between the experimental groups. */$ p.value < 0.05; **p.value < 0.01; *** p.value < 0.001. Error bars indicate s.e.m.
