Fig. 2
Effects of NF-YA inactivation in tumor prostate cells. A Expression of NF-YA splice variants in prostate epithelial normal and cancer cells by western blot analysis of total cellular extracts. N1 = primary healthy epithelial prostate cells, C10, C17 = primary BPH prostate cells. RWPE-1, PNT1A = normal immortalized cell lines. PC3, DU145, LNCaP = PCa cell lines. Tubulin was used as loading control of total cellular extracts. B Expression levels of NF-YA isoforms quantified by RT-qPCR and reported as fold change of NF-YAs/NF-YAl mRNA ratio vs N1 cell line levels, arbitrarily set at 1. Rpl21 was used as reference gene. C Western blot analysis of NF-YA expression in PC3 cells untreated (CTR) or infected with scramble (shCTR) or NF-YA-targeting shRNA (shNF-YA). Tubulin was used as loading control. D Colony number of shCTR and shNF-YA cells cultured in anchorage-independent growth conditions. Data represent mean ± SEM (unpaired t-test: ***p < 0.001, n = 4). E Percentage of cell migration of shCTR and shNF-YA cells measured by transwell assay. Data represent mean ± SEM (unpaired t-test: *p < 0.05, n = 3). F Percentage of cell invasion of shCTR and shNF-YA cells measured by transwell assay. Data represent mean ± SEM (unpaired t-test: **p < 0.01, n = 2). G Representation of tumor incidence after 5 weeks from s.c. injection of shCTR and shNF-YA PC3 cells into SCID Hairless Outbred (SHO®) mice. H (Left panel) Volumes (mm3) of shCTR and shNF-YA xenografted tumors at the indicated time points. Data represent mean ± SEM (two-way ANOVA with Holm-Sidak’s test: **p < 0.01, ***p < 0.001, ****p < 0.0001, n = 7). (Right panel) Tumor weight of xenografted tumors isolated after 5 weeks from cell inoculation. Data represent mean ± SEM (unpaired t-test: **p < 0.01)