γ-IFN–induced increase of surface MHC class I expression is not affected by PML-specific RNA interference or by expression of PML mut ex3.

HEK293 cells were either untreated (left panel) or treated with 1000 U/ml γ-IFN (right panel) (time [t] = 0), and subsequently transfected with PML-specific siRNA duplex (middle row) or with GFP-PML mut ex3 (bottom row) (t = 6 hours). After 2 additional days (t = 54 hours), cells were processed for fluorescence microscopy and flow cytometry. Cells growing on glass coverslips were immunostained with anti-PML followed by GAM-Alexa 594 antibodies (red fluorescence, first and fourth columns) and counterstained with DAPI (blue fluorescence, second and fifth columns). Cells processed for flow cytometry were immunostained with anti–MHC class I mAb (W6.32) followed by GAM-PE. Histograms of MHC class I expression are shown for control and siRNA-transfected cell cultures, while bivariate dot plots of GFP fluorescence (x-axis) and MHC class I expression (y-axis) are reported for PML mut ex3–transfected cell cultures. Percentage of GFP-positive cells (ie, of PML mut ex3–transfected cells) is reported. The negative control of MHC class I expression, obtained using isotype-matched antibodies followed by GAM-PE, is shown in gray.