Figure 3

MeCP2 functional ablation determines alteration of NE components expression. (A) PC-3, LNCaP and NIH-3 T3 cells were transfected with siRNA CTRL and siRNA MeCP2 oligos. Protein extracts were prepared at 5 and 7 days after transfection. Western blot assays against MeCP2 (70 kDa), lamin A/C (69 kDa, 62 kDa respectively), lamin B1 (68 kDa), LBR (58 kDa) and GAPDH (34 kDa), as loading control, were performed. (B) Quantitative RT-PCR showed that 7 days of MeCP2 silencing in PC-3 cells causes alteration of mRNA levels of LMNA, lamin B1 and LBR genes. mRNAs levels were normalized with respect to GAPDH housekeeping gene and were expressed as arbitrary units (statistics were performed using Student’s test. *: significant difference between siRNA CTRL and siRNA MeCP2, P < 0.001; °, significant difference between siRNA CTRL and siRNA MeCP2, P < 0.05). (C) Intracellular localization of endogenous lamin A and MeCP2 proteins. Fixed 7day MeCP2 silenced PC-3 cells and control cells were treated with antibodies against MeCP2 (green) and lamin A (red) proteins and analysed by confocal microscopy. Low levels of MeCP2 protein do not alter lamin A distribution, however an irregular nuclear rim is observed (C3-C6; C3a-C6a fluorescent and phase contrast images respectively). (D) In the line diagram is shown the local intensity distribution (diagonal white lines through the images) of MeCP2 (green), lamin A (red) in box1 (siRNA CTRL cell) and in box2 (siRNA MeCP2) respectively. (E) In the line diagram is shown the local intensity distribution of MeCP2 (green) and lamin A (red) of cell in box3.