Figure 6.

Overexpression of DAXX triggers formation of large PML-NBs together with an accumulation of HP1 proteins in these nuclear bodies. (A) Western blot analysis of total cell extracts from MRC5 cells transduced with an empty vector or a vector expressing Myc-DAXX. Membranes were probed for Myc to verify expression of the transduced proteins, and for DAXX. α-Tubulin is a loading control. M, molecular weight marker. (B) Fluorescent microscopy visualization of PML (green) and DAXX (red) in MRC5 cells treated as in (A) shows formation of large PML-NBs upon Myc-DAXX overexpression (white arrowheads). Scale bar is 10 μm. (C) Fluorescent microscopy visualization of nascent global RNA transcription in vivo in MRC5 cells empty or overexpressing Myc-DAXX. Nascent RNAs were labeled with 5-ethynyl uridine (EU) for 3 h and revealed by the Click-iT chemistry (red). Co-staining with Myc (green) and PML (cyan, pseudo-color) shows absence of transcription within the large PML-NBs upon overexpression of DAXX. Insets represent enlarged images (3×) of selected area. Scale bar is 10 μm. (D and E) Fluorescent microscopy visualization of Myc-DAXX (Myc, green) and HP1γ or H3K9me3 (red) in MRC5 cells treated as in (A) shows accumulation of HP1γ (D) in absence of H3K9me3 (E) in the large PML-NBs upon overexpression of Myc-DAXX (white arrowheads). Scale bar is 10 μm.