Formation of radiation-induced protein foci is not affected by depletion of Jarid1A.

<p>Detection of repair foci in scr or Jarid1A A1+A3 siRNA transfected HeLa cells after ion microirradiation. 72 h after transfection cells were irradiated in a small angle (10°) with 55 MeV carbon ions. After incubation, cells were fixed and indirect immunofluorescence was performed to detect the foci formation of γH2AX and BRCA1 (A), Rad51 and γH2AX (B), or 53BP1 and γH2AX (C). For each sample, two typical cells are depicted. For quantitative evaluation, cells with γH2AX tracks were divided in 3 groups, depending on whether the protein in question formed foci that overlapped with γH2AX foci along the whole track, or occasional foci that overlapped with some of the γH2AX foci in the track, or no overlapping foci. Indicated are means (+/- SEM) from at least 50 evaluated cells.</p

Immunoprecipitation performance and affinity of the PARP1 nanobody.

<p>(A) Immunoprecipitation of endogenous hPARP1 from whole-cell lysates of HEK293T cells with PARP1 nanotrap. Input (I), flow-through (FT) and bound (B) fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining (left) and western blotting with anti-PARP1 antibody (right). (B) Affinity measurement of the PARP1 nanobody with Biacore SPR. The sensorgrams for the nanobody at different concentrations of hPARP1 are indicated.</p

Jarid1A depletion does not affect cell proliferation.

<p>(A) No difference in the cell number 72 h after transfection with scr or Jarid1A siRNA. In all experiments involving protein extracts, the cell number of the different transfection samples was determined prior to protein extraction. Indicated are mean cell numbers (+/- SD) of HeLa cells (12 experiments), U2OS cells (3 experiments) and MCF-7 cells (2 experiments), each normalized with respect to the untransfected control samples. (B) Consistent cell cycle distribution after down-regulation of Jarid1A. 72 h after siRNA transfection HeLa cells were harvested, stained with PI and analyzed by flow cytometry. (C) Constant plating efficiency of the transfected cells. After incubating transfected and control HeLa cells for 10 days, colonies were stained with methylene blue and the number of colonies was determined. Graph indicates the mean plating efficiency (+/- SD) of 3 independent colony formation assays. (D) Methylene blue stained colonies 10 days after seeding of 300 cells per well.</p

Intracellular F2H analysis of the PARP1 chromobody.

<p>BHK-F2H cells were pairwise co-transfected with the PARP1 chromobody fused to TagRFP and one of the GFP-tagged bait constructs: GFP alone, GFP-hPARP1, GFP-hPARP2, GFP-hPARP3, GFP-hPARP9, wild-type ZnF2-GFP and ZnF2mut-GFP. The cells were fixed, stained with DAPI and subjected to fluorescence microscopy. Upper row, green channel: GFP-fusion proteins are enriched at the “spot” in the nuclei of transfected BHK-F2H cells (arrows). Middle row, red channel: binding of the PARP1 chromobody to the full-length GFP-hPARP1 and to the wild-type ZnF2-GFP is visible as local enrichments of the red fluorescent signals (arrows). Neither interaction of the PARP1 chromobody with hPARP2, 3, or 9, nor interaction with the mutant ZnF2mut-GFP construct (G161T, A188S and T189A) can be observed. Co-transfection with GFP (first column) served as negative control to exclude non-specific binding of the PARP1 chromobody to GFP. Scale bar, 5 μm.</p

Determination of the PARP family selectivity and species reactivity of the PARP1 nanobody.

<p>(A) Immunoprecipitation of GFP-tagged hPARP1 (141 kDa), hPARP2 (93 kDa), hPARP3 (87 kDa), hPARP9 (123 kDa) and GFP (27 kDa, negative control) with the PARP1 nanotrap from transiently transfected HEK293T cells. RFP-Trap was used as control. Input (I), flow-through (FT) and bound (B) fractions were separated by SDS-PAGE followed by immunoblotting with anti-GFP antibody. (B) Immunoprecipitation of endogenous PARP1 from mouse (MEF) and hamster (BHK) cells with the PARP1 nanotrap. The fractions were analyzed by SDS-PAGE and immunoblotting with anti-PARP1 antibody.</p

Epitope mapping of the PARP1 nanobody by immunoprecipitation of hPARP1 domains.

<p>(A) Schematics depicts hPARP1 domain structure: DNA-binding domain (45 kDa), automodification domain (19 kDa) and catalytic domain (58 kDa). Purified recombinant hPARP1 domains were subjected to immunoprecipitation with the PARP1 nanotrap Input (I), flow through (FT) and bound (B) fractions were analyzed by SDS-PAGE followed by Coomassie Blue staining. (B) GFP- or mCherry-tagged hPARP1 domains were transiently expressed in HEK293T cells: full DNA-binding domain (DBD), DBD constituting zinc fingers (ZnF1, ZnF2, ZnF3), as well as the WGR domain (part of the catalytic domain). The cells were lysed and immunoprecipitated with the PARP1 nanotrap and RFP-Trap or GFP-Trap as control. The fractions were subjected to SDS-PAGE followed by immunoblotting with anti-GFP or anti-RFP antibody.</p

Residual γH2AX foci and reporter plasmid-based DSB repair events are not affected by depletion of Jarid1A.

<p>A) Mean background and residual foci number (+/- SEM) in at least 20 cells per sample after 5 Gy X-rays. Cells were fixed before and 24 h or 48 h after irradiation and indirect immunofluorescence was performed. Semi-automatic detection and characterization of spontaneous and residual γH2AX foci in the cells was performed by using the PlugIn FociPicker3D [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0156599#pone.0156599.ref028" target="_blank">28</a>]. B) Relative number of GFP expressing cells, normalized to the frequency in untransfected HeLa pEJ (left panel) and pGC (right panel) control cells, after transfection of I-SceI expression plasmid into untreated cells and cells treated with scr siRNA or Jarid1A A1+A3 siRNA. Indicated are means ± SEM of 5 independent experiments.</p

Efficient down-regulation of Jarid1A is associated with global increase of H3K4me3.

<p>(A) Decreased levels of Jarid1A in whole cell protein extracts of HeLa cells after siRNA transfection. Western blot images show levels of Jarid1A in untransfected cells (cont), in cells transfected with scrambled siRNA (scr) and in cells transfected with Jarid1A A1+A3 siRNA. Normalized average amount (+/- SD) of Jarid1A protein after siRNA transfection of HeLa cells was determined by quantitative analysis of Western blots of protein extracts obtained in 10 independent experiments. (B) Decreased Jarid1A and increased H3K4me3 signal intensity after indirect immunofluorescence in HeLa cells transfected with Jarid1A A1+A3 siRNA. Microscopic images were obtained at comparable exposure times and display the cell nuclei stained with DAPI in blue, the protein Jarid1A in red and the histone modification H3K4me3 in green. The graph indicates normalized average fluorescence of Jarid1A and H3K4me3 in nuclei of 20 randomly chosen cells. (C) Increase of H3K4me3 in whole cell protein extracts of HeLa cells after depletion of Jarid1A. Western blot images show levels of Jarid1A and H3K4me3. Average levels of H3K4me3 (+/- SD) were determined by quantitative analysis of protein extracts obtained in 3 independent experiments via Western Blot.</p

Depletion of Jarid1A results in histone hyperacetylation.

<p>(A) Increase of H4K16ac, H3K9ac, and H3K56ac in whole cell protein extracts of HeLa cells after siRNA transfection. Western blot images show levels of the histone modifications in cells transfected with scrambled siRNA (scr) and Jarid1A A1+A3 siRNA. Graphs indicate the means (+/- SD) from 5 independent experiments for H4K16ac and 3 independent experiments for H3K9ac and H3K56ac after quantitative analysis of Western blots. The effect of H4K16ac is statistically significant (p = 0.0035). (B) The amounts of the histones H3 and H4 are not affected by down-regulation of Jarid1A. Indicated are means (+/- SD) from 3 experiments.</p

Depletion of Jarid1A enhances radiosensitivity.

<p>Survival fraction of the differently treated HeLa cells irradiated at 72 h after siRNA transfection. Cells were irradiated with 0 Gy, 2 Gy 5 Gy or 10 Gy X-rays and incubated for 10 days before fixation and methylene blue staining of colonies. For every dose the mean value of the cell survival (+/- SD) of 3 independent colony forming assays is shown. Data were fitted with a linear-quadratic model and statistical significance was determined by F test. After Jarid1A depletion, survival is significantly reduced as compared to scr siRNA transfected cells (p = 0.0025) and untransfected controls (p < 0.0001).</p