Fig. 3

Effects of stable overexpression of NF-YA isoforms in tumor cells. A Western blot analysis of total extracts from PC3 cells stably infected with Empty, NF-YAl and NF-YAs lentiviral particles. Tubulin was used as loading control. B, C Colony number of Empty, NF-YAl and NF-YAs stable cell lines in anchorage-dependent and anchorage-independent conditions, respectively. Data represent mean ± SEM (one-way ANOVA with Fisher’s LSD test: *p < 0.05, **p < 0.01, ***p < 0.001, ns, not significant, n = 6). D Size of Empty, NF-YAl and NF-YAs MTSs calculated as projected area at the indicated time points. Data represent mean ± SEM (two-way ANOVA with Holm-Sidak’s test: *p < 0.05, **p < 0.01, ***p < 0.001, ****p < 0.0001, n between 8 and 68 spheroids from 8 independent experiments). E Representative immunofluorescence images of Ki67/Dapi and Calcein-AM/PI staining performed on Empty, NF-YAl and NF-YAs MTSs. Scale bar = 500 μm. The lower panel shows details of Calcein-AM/PI staining by confocal microscopy. Scale bar = 50 μm. F Percentage of BrdU-positive cells following incubation of MTSs with 20 μM BrdU for 16 h. Data represent mean ± SEM (one-way ANOVA with Fisher’s LSD test: **p < 0.01, ****p < 0.0001, n = 4). G Percentage of AnnexinV-positive cells identified by cytofluorimetric analysis of MTSs. Data represent mean ± SEM (one-way ANOVA with Fisher’s LSD test: *p < 0.05, ns, not significant, n = 3). H Optical microscopy images representative of Empty, NF-YAs and NF-YAl MTSs morphology. Scale bar 4X = 500 μm, scale bar 10X = 250 μm (I) Representative images of H&E stained sections of MTSs. Scale bar = 100 μm