PML mut ex3 induces cytoplasmic localization of PML molecules and does not affect MHC class I expression.

(A) Cells growing on glass coverslips were either untreated (top row) or transfected with PML mut ex3 (bottom row) by the Lipofectamine 2000 procedure. At 2 days after transfection, cells were stained with anti-PML mAb followed by GAM-Alexa 594 (red) and counterstained with DAPI (blue). Localization of PML molecules in relation with the cell nucleus was achieved by merging the 2 fluorescence pictures. The amino-acid sequences of the PML mut ex3 protein and of the murine PML mutant F12dG are compared. Both mutants arise from a point deletion in exon 3 of the PML gene that causes a frameshift and translational termination of PML proteins at exon 3. The 16-aa string underlined at the C-terminus of both proteins corresponds to the sequence that derives from the mutation-induced frameshift and that is not present in wt PML. (B) ACN cells were transfected with GFP-tagged PML mut ex3. At 2 days after transfection, cells were processed for microscopic and flow cytometric analyses. Cells growing on glass coverslips were immunostained with GAM-Alexa 594 (middle column) either alone (top row) or preceded by anti-PML mAb (bottom row) and counterstained with DAPI (right column). GFP fluorescence is shown in the left column. Of note is the presence of one untransfected cell that was negative for GFP and displayed a PML pattern typical of NBs after immunostaining with anti-PML mAb. Cells processed for flow cytometry were immunostained with anti–MHC class I mAb (W6.32) followed by GAM-PE. In the bivariate dot plot of GFP (x-axis, green fluorescence) versus MHC class I (y-axis, red fluorescence), the percentage of GFP-positive cells (ie, of transfected cells) is reported. The negative control for MHC class I expression, obtained using isotype-matched antibodies followed by GAM-PE, is shown in gray.