FIG.5.

Overexpression of the PARP-1 binding domain of WRN strongly reduces PAR polymer formation after H₂O₂ treatment. TERT-1604 cells were transfected with either pEGFP-WRN (aa 949 to 1092) (A) or pEGFP alone (B). Thirty-six hours posttransfection, the cells were synchronized (early S phase) with HU for an additional 18 h. The cells were then incubated with H₂O₂ (500 μM) for 10 min at 37°C. After fixation, the cells were probed with anti-PAR antibodies. Arrowheads, EGFP-WRN- (aa 949 to 1092) and EGFP-transfected cells; arrow (A, g), one cell with low EGFP-WRN (aa 949 to 1092) expression. Bars (A, i, and B, f), 10 μm. Pictures correspond to representative transfected cells showing high-level of expression either of EGFP-WRN (aa 949 to 1092) (A) or EGFP alone (B). Approximately 80% (33 of 40) of the EGFP-WRN RQC-overexpressing cells showed less PAR polymer formation than nontransfected cells.