MHC class I expression is not affected by PML-specific RNA interference.

HeLa cells were transfected with PML-specific siRNA duplex (second row), with GFP-PML (third row), or cotransfected with siRNA duplex and GFP-PML (fourth row) using the Oligofectamine procedure. Control cells in the first row were treated with Oligofectamine alone. At 48 hours after transfection, cells were processed for fluorescence microscopy and flow cytometry. Cells growing on glass coverslips were immunostained with anti-PML mAb followed by donkey antimouse FITC and anti-SP100 mAb followed by donkey antigoat Cy3 (control and siRNA duplex-treated samples, first 2 rows), or with anti-PML mAb only, followed by GAM-Alexa 594 (samples transfected with GFP-PML, last 2 rows). The green fluorescence is shown in the first column, the red fluorescence in the second, and the blue fluorescence from DAPI in the third. In the fourth row, a microscopic field comprising one of the few GFP-positive cells was chosen, which presumably was not transfected with siRNA but only with GFP-PML, to emphasize the disappearance of PML-containing NBs induced by RNA interference. Cells processed for flow cytometry were immunostained with anti–MHC class I mAb (W6.32) followed by GAM-PE. Data are reported in the fourth column as histograms of MHC class I expression (top 2 rows, negative control provided by isotype-matched mAb indicated in gray) and bivariate dot plots (bottom 2 rows) of GFP fluorescence (x-axis) and MHC class I expression (y-axis).