Figure 2
MeCP2 silencing determines PC-3 cells suffering without triggering a severe apoptotic and senescence effects. (A) Fixed PC-3 cells, transfected with siRNA CTRL or siRNA MeCP2 oligos were analyzed by confocal microscopy. Phase contrast representative pictures are shown. (B)Annexin V-FITC/PI FACS analysis was performed in PC-3 cells after 5 and 7 days of MeCP2 RNAi. This staining reveals the amount of early apoptosis (LR = A+ + PI-), late apoptosis (UR = A+ + PI+), living cells (LL = A- + PI-) and necrotic cells (UL = A+ + PI+); x-axis Annexin V stain signal, y-axis PI stain signal. The percentage of apoptotic cells (UR + LR) between control and siMeCP2 samples of different experiments is shown in AnnexinV positive cells bar chart (statistic were performed using ANOVA. *: significant difference between siRNA CTRL and siRNA MeCP2 PC-3 cells after 5 days of silencing, P < 0.01. **: significant difference between siRNA CTRL and siRNA MeCP2 PC-3 cells after 7 days of silencing, P < 0.001). (C) PC-3 cells were transfected with siRNA CTRL and siRNA MeCP2 oligos. After 7 days of transfection protein extracts were prepared and western blot against Cleaved-PARP (Asp214 89 kDa) and GAPDH, as loading control, was performed. (D) SA-β-galactosidase assay was performed at 7 days after the first MeCP2 silencing. Blue cells in (D1) untreated, (D2) siCTRL and (D3) siMeCP2 cells are shown. Bar chart shown the percentage of senescent PC-3 cells after 7 days of siRNA CTRL (0.23%) and siRNA MeCP2 (4.4%) calculated by the ratio between the number of senescent and non-senescent cells in different microscope fields (data are means ± SD bars). (E) Identification of SAHFs (senescence-associated heterochromatic foci). Fixed 7day MeCP2 silenced PC-3 cells and control cells were treated with antibodies against HP1γ (green) and H3K9me3 (red) proteins and stained with DAPI. Cells were analysed using fluorescence microscope and an enlargement of each representative senescence stain (indicated by the white arrow) is showed (box a, box b and box c). In bar chart is indicated the percentage of senescent PC-3 cells after 7 days of siRNA CTRL (1%ca) and siRNA MeCP2 (4%ca) calculated by the ratio between the number of SAHFs and total cells in different microscope fields (data are means ± SD bars).