Fig. 5: PARP1 is the writer of DNA damage dependent mono-Asp/Glu-ADPr.

A HPF1KO U2OS cells were treated with 1 µM Olaparib for 30 min to inhibit PARP1/2 or with DMSO control, followed by 2 mM H₂O₂ for 10 min as indicated to induce DNA damage, then harvested and lysed according to our optimized procedure, immunoblotted with the indicated antibodies and stained with Ponceau S to show total protein load. B WT U2OS cells were treated with 1 µM Olaparib for 30 min to inhibit PARP1/2, 10 nM AZD5303 or AZD9475 for 30 min to selectively inhibit PARP1, or with DMSO control, followed by 2 mM H₂O₂ for 10 min as indicated to induce DNA damage, then harvested and lysed according to our optimized procedure, immunoblotted with the indicated antibodies and stained with Ponceau S to show total protein load. C Top: immunofluorescence quantification of WT U2OS cells treated as indicated, then fixed with methanol and stained with AbD43647 IgG and DAPI. The signal intensity of ~100 cells from a representative of 2 independent experiments is shown. The red bars indicate sample mean. Bottom: representative confocal images. Scale bar = 10 µm. D HPF1KO U2OS cells were processed and immunoblotted as indicated. E Top: immunofluorescence quantification of HPF1KO U2OS cells processed as indicated. The signal intensity of ~100 cells from a representative of 2 independent experiments is shown. The red bars indicate sample mean. Bottom: representative confocal images. Scale bar = 10 µm. For figures (A–C) shown is a representative result from three independent experiments.