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.</p

Differential degradation of PML isoforms in response to As₂O₃.

<p>(A) U373MG cells stably expressing each PML isoform were untreated (C) or treated with 5 µM of As₂O₃ for the indicated time (24 h or 48 h). (B) U373MG cells stably expressing PMLVI, PMLVII or PMLIII-SIM were untreated (C) or treated from 24 to 48 h with 5 μM of As₂O₃. The different cell extracts were analyzed by Western blot with anti-PML or anti-actin antibodies. (C) U373MG cells were untreated (C, lane 1) or treated with 1000 units/ml of IFNγ (lanes 2 and 4) to increase the expression of the endogenous forms of PML. One day later, As₂O₃ was added at the concentration of 5 μM (lanes 3 and 4) for 24 h. In A, B and C, cell extracts were analyzed by Western blot with anti-PML (top) or anti-actin antibodies (bottom). Unmodified PML isoforms are indicated by arrowheads and PML forms resistant to As₂O₃-induced degradation in IFN-treated cells by stars.</p

The β regulatory subunit of the 11S proteasome was recruited to PML NBs in response to As₂O₃ in cells stably expressing PMLI, PMLII, PMLIV and PMLV.

<p>Confocal immunofluorescence analysis of PML and 11Sβ were performed on U373MG cells stably expressing PMLI, PMLII, PMLIV or PMLV, treated or not with 5 μM of As₂O₃ for 1 h. PML and 11Sβ were detected with a mouse anti-PML and a rabbit anti-11Sβ antibodies followed by the corresponding anti-IgG antibody conjugated to Alexa 594 (red) and 488 (green), respectively. The merged images revealed co-localization of PML and endogenous 11Sβ for PMLI, PMLII, PMLIV and PMLV in the presence of As₂O₃ in nearly all cells.</p

Detection by BRET of the increase in the SUMOylation of nuclear PML isoforms by SUMO1 in response to As₂O₃ in living cells.

<p>HEK293T cells, transiently transfected with a fixed amount of a Luc-PML fusion (PMLI to VII) and increasing amounts of YFP-SUMO1, were treated in the presence or absence of 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).The BRET donor of energy (luciferase fused to a PML isoform) and acceptor of energy (YFP fused to SUMO) are illustrated; upon addition of the membrane permeant luciferase substrate (coelenterazine Deep Blue), the bioluminescent signal resulting from the degradation of the substrate (emission 480 nm) is transferred to the YFP acceptor when the acceptor is within close proximity (50–100A°). The transferred energy results in a fluorescent signal emitted by the YFP acceptor (emission 530 nm). The BRET signal corresponds to the acceptor fluorescence/donor bioluminescence ratio (y axis) and is plotted as a function of the YFP/Luc fusion protein expression (x axis). ‘K’ represents a lysine to which SUMO can be covalently coupled.</p

The SIM of PML is not required for the interaction with RNF4 but is necessary for PML ubiquitination in response to As₂O₃.

<p>(A) Co-immunoprecipitation of RNF4 and PML isoforms and mutant. HEK293 cells were co-tranfected with a construct encoding Flag-RNF4 and PMLIII, PMLVI or PMLIII-SIM. As controls, cells were transfected with PMLIII or RNF4 alone. Two days later, the different cell extracts were immunoprecipitated with an anti-Flag antibody as described in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0044949#s4" target="_blank">Materials and Methods</a> and samples were analyzed by Western blot using anti-PML and anti-Flag antibodies. (B) Ubiquitination of PML in response to As₂O₃. PML^−/− MEFs were transiently transfected with the empty vector (EV) or PMLIII (left panel) or contructs encoding PMLIII, PMLIII-SIM or PMLVI (right panel). Two days later, they were treated with As₂O₃ for 1 h. The cell extracts were immunoprecipitated with rabbit anti-PML antibody and the immunoprecipitate was analyzed by Western blot using anti-ubiquitin and anti-PML antibodies.</p

Degradation of PML isoforms in the presence of RNF4 and/or As₂O₃.

<p>U373MG cells were transiently co-transfected with each of the different PML isoforms (PMLI to VII) and RNF4 (ratio PML/RNF4∶1/1). One day post-transfection, cells were treated with 5 μM for 6 h. The different cell extracts were analyzed by Western blot with anti-PML, anti-Flag (Flag-RNF4) or anti-actin antibodies. Unmodified PML isoforms are indicated by arrowheads. For each PML isoform, samples in the presence (lanes 1–2) and in the absence of RNF4 (lanes 3–4) were loaded on the same gel to allow comparison of the protein levels.</p

Requirement of the SIM for the recruitment of theβ regulatory subunit of the 11S proteasome to PML NBs in response to As₂O₃.

<p>Confocal immunofluorescence analysis of PML and of the β regulatory subunit of the 11S proteasome (11Sβ) were performed on U373MG cells stably expressing PMLIII (A) or PMLIII-SIM (B), treated or not with 5 μM of As₂O₃ for 1 h, 24 h or 48 h. PML and 11Sβ were detected with a mouse anti-PML and a rabbit anti-11Sβ antibodies followed by the corresponding anti-IgG antibody conjugated to Alexa 594 (red) and 488 (green), respectively. The merged images at 1 h of As₂O₃ treatment revealed in all cells a co-localization of endogenous 11Sβ with PMLIII but not with PMLIII-SIM mutant.</p

PMLVI was unable to recruit the β regulatory subunit of the 11S proteasome to PML NBs in response to As₂O₃.

<p>Confocal immunofluorescence analysis of PML and11Sβ were performed on U373MG cells stably expressing PMLVI treated or not with 5 μM of As₂O₃ for 1 h, 24 h or 48 h. PML and 11Sβ were detected with a mouse anti-PML and a rabbit anti-11Sβ antibodies followed by the corresponding anti-IgG antibody conjugated to Alexa 594 (red) and 488 (green), respectively.</p