<p>(A) Nucleolar localization of PML. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) or empty vector (c) were treated with 5 µM MG132 for 16 h. Endogenous TTRAP (green), PML (red) and NPM (blue) localization was analyzed by triple immunofluorescence. Images are representatives of two separate experiments performed on two independent clones for each cell line. (B) Quantification of PML nucleolar localization. Cells were treated as in A. 200 cells from two independent experiments were counted and scored for PML in the nucleolus (*, p<0.05). (C) Mutant DJ-1 L166P does not affect levels of endogenous PML. SH-SY5Y cells stably expressing wt DJ-1 (W), L166P (L) or empty vector (c) were treated with 5 µM MG132 for 16 h or left untreated, as indicated. The expression of endogenous PML and overexpressed FLAG-DJ-1 were measured with anti-PML and anti-FLAG antibodies, respectively. Beta-actin was detected as loading control. (D) Quantification of PML levels. Densitometric analysis of protein bands was performed on two independent experiments. Relative expression of endogenous PML was normalized to beta-actin. Data are expressed as the percentage of untreated condition in empty cells (c). (E) Nucleolar localization of p53. Cells were exactly as in A. Localization of endogenous p53 (green), NPM (blue) and FLAG-DJ-1 (red) were analyzed by triple immunofluorescence. Bars, 10 µm. (F) Quantification of p53 nucleolar localization. Analysis was performed as in B (NS, non significant).</p
