Interaction by BRET of RNF4 with SUMOylated PMLIII or PMLVI.

<p>HEK293T cells transiently transfected with a Luc-PML fusion (PMLIII or VI) and increasing amounts of RNF4-YFP, were untreated or treated with As₂O₃ (5 µM, 4 h) and used for BRET titration assays. BRET saturation curves are presented for each PML isoform in the absence (open square) or presence of As₂O₃ (closed square). Bar graphs are for presenting the BRET signal (BRET), the luciferase expression and YFP expression of individual untreated (gray bars) or treated (black bars) samples. A strong increase in the YFP/Luc expression ratios upon As₂O₃ treatment was observed for the BRET pair Luc-PMLIII/RNF4-YFP when compared to the YFP/Luc ratios obtained with the same untreated samples. This increase in the YFP/Luc expression ratios was accounted by a significant decrease in the level of Luc-PMLIII in the presence of As₂O₃ whereas the level of RNF4-YFP remained essentially unaffected by the treatment; this is evidenced by the bar graphs of the treated and untreated samples presented below the BRET curves. The shift toward higher YFP/Luc ratios resulting from Luc-PML degradation was much more limited for PMLVI than for the other nuclear PML isoforms such as PMLIII (this figure) and PMLI to PMLV (<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044949#pone.0044949.s004" target="_blank">Figs. S4A and S4B</a>). Noticeably, in response to As₂O₃, the Luc-PML reduced expression was not found to be dependent on the concentration of RNF4 added. Since this assay is conducted in conditions of overexpression of both PML and RNF4 in transiently transfected cells, we believe that this is possibly due to the fact that, in comparison with PML and RNF4, the ubiquitin and/or proteasomal machineries were limiting in the cells.