MeCP2 overexpression diminishes MHC class I expression.

<p>N2A cells transfected with pCMX vectors expressing either murine or human MeCP2 were immunostained for one of the three MHC class I molecules (K^k, L^d or D^d), β2-microglobulin or the transferrin receptor on the cell surface as well as for intracellular MeCP2. The level of staining was then analysed by fluorescence-activated cell sorting. Panel A: Dot plots of the amount of surface antigen (x-axis) against the amount of intracellular MeCP2 (y-axis) in cells transiently transfected with pCMX expressing human MeCP2 and analysed 48 hrs later. Panel B: For each kind of staining, the variation in expression level was calculated as the ratio of MFI of MeCP2 overexpressing cells over MFI of mock-transfected cells. The histograms summarise the mean (±SEM) of the variation in cell surface levels from 15 independent transfections with vectors expressing mouse MeCP2<i>A</i> (grey fill) and 12 independent transfections with vectors expressing human MeCP2<i>A</i> (black fill). Panel C: N2A and NIH3T3 cells were transfected with empty pcDNA3.1(+) (mock cells) or expressing Myc-tagged human MeCP2<i>A</i> or <i>B</i> isoforms. 48 h after transfection, cells were subjected to double staining against cell surface MHC class I molecules and intracellular Myc-tagged MeCP2, then analyzed by flow cytometry. The variation in expression level of MHC class I molecules was calculated as the ratio of MFI of MeCP2 over-expressing cells over the MFI of mock cells. The histograms represent the mean (±SEM) of the cell surface level variation from 4 independent transfections with each of the vectors. Panel D: A representative example of dot-plots obtained for double immunostaining of transiently transfected N2A cells with anti-Myc 9E10 and rat-anti-mouse-MHC I M1/42 monoclonal antibodies. Dotted and continuous circles indicate the different populations expressing high and intermediate levels of MeCP2, respectively. Statistical significance of difference between groups was analysed by using an unpaired t-test (**, <i>p</i><0.01 ; ***, <i>p</i><0.001).