TGFβ induces formation of endogenous complexes between Smads and PARP-1/2 in HaCaT cells.

<p>(<b>a</b>) Immunoprecipitation of Flag-Smad2/3/4 followed by immunoblotting for PARP-1 and PARP-2 in cell lysates of transiently transfected HEK-293T cells with the indicated plasmids and after stimulation with vehicle (-TGFβ) or 5 ng/ml TGFβ1 for 30 min. Expression levels of all transfected proteins and endogenous PARP-1 and PARP-2 are shown in the total cell lysate (TCL) immunoblot of the HEK 293T cells. PARP-1 immunoblot also serves as protein loading control. Stars mark non-specific protein bands. (<b>b</b>) Immunoprecipitation of Smad2/3 followed by immunoblotting for PARP-1, PARP-2, Smad2/3 and Smad4 in HaCaT cells stimulated with vehicle (-TGFβ) or with 5 ng/ml TGFβ1 for 30 min. Negative control immunoprecipitation using non-specific IgG is shown. TCL shows the levels of endogenous proteins before immunoprecipitation. PARP-1 immunoblot also serves as protein loading control and C-terminal phospho-Smad2 (p-Smad2) serves as control for the efficiency of stimulation of TGFβ signaling. (<b>c</b>) Immunoprecipitation of Smad2/3 followed by immunoblotting for PARP-1, PARP-2, Smad2/3 and Smad4 in HaCaT cells transfected with the indicated siRNAs and stimulated with 5 ng/ml TGFβ1 for 30 min or not (-TGFβ). Efficiency of knockdown of PARP-1 and PARP-2, total Smad levels, phospho-Smad2 levels and protein loading (α-tubulin) controls can be seen in the TCL. (<b>d</b>) In vitro PARylation assay after glutathion-pulldown of control GST protein or GST-Smad3, truncated mutant of GST-Smad3 (ΔMH2) and GST-Smad4 in the presence of recombinant PARP-1 and/or recombinant PARP-2 as indicated. A star (weak signal) indicates the position of PARP-2 in addition to the arrow. A longer exposure of the autoradiogram around the migrating position of PARP-2 is shown at the bottom. Note the position of ADP-ribosylated Smad proteins that migrate at the size of the core non-ADP-ribosylated proteins. The input amounts of recombinant proteins were calculated based on staining of test SDS-PAGE with CBB as shown in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone.0103651.s001" target="_blank">Fig. S1</a>. The figure shows results from representative experiments that were repeated at least twice.</p

PARG forms complexes with Smad proteins and de-ADP-ribosylates Smad3.

<p>(<b>a</b>) Immunoprecipitation of Flag-Smad2, Flag-Smad3 or Flag-Smad4 followed by immunoblotting for myc-PARG in cell lysates of transiently transfected 293T cells with the indicated plasmids and after stimulation with vehicle (-TGFβ, left panel) or 5 ng/ml TGFβ1 for 30 min (right panel). Expression levels of all transfected proteins are shown in the TCL immunoblot of the 293T cells. (<b>b</b>) Immunoprecipitation of Flag-Smad2/3/4 followed by immunoblotting for myc-PARG in cell lysates of transiently transfected 293T cells with the indicated plasmids and in the absence of stimulation with TGFβ. Expression levels of all transfected proteins are shown in the TCL immunoblot of the 293T cells. α-Tubulin immunoblot serves as protein loading control. Stars mark non-specific protein bands. (<b>c</b>) Immunoprecipitation of endogenous Smad2/3 followed by immunoblotting for transfected myc-PARG in 293T cells stimulated with vehicle (-TGFβ) or with 5 ng/ml TGFβ1 for 30 min. Negative control immunoprecipitation using non-specific IgG is shown. TCL shows the levels of endogenous Smad2/3 proteins and transfected myc-PARG before immunoprecipitation. Smad2/3 immunoblot also serves as protein loading control. (<b>d</b>) In vitro de-ADP-ribosylation assay of Smad3 using PARG. GST-Smad3 was first ADP-ribosylated using recombinant PARP-1. The proteins were pulled-down and washed, prior to reconstitution with PARG reaction buffer and increasing amounts of recombinant PARG (shown as milli-units (mU) of enzymatic activity). The ADP-ribosylated proteins are shown in the autoradiogram along with the CBB-stained input GST-Smad3 levels. Panels a–c show results from representative experiments that were repeated at least twice and panel d shows results from representative experiments that were repeated at least three times.</p

PLA of endogenous Smad3 and PARP-1 complexes in HaCaT cells.

<p>(<b>a</b>) HaCaT cells were analyzed with PLA using antibodies against Smad3 and PARP-1 after transfection with control or the indicated specific siRNAs and stimulation with vehicle (-TGFβ) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. Cells stimulated with 10 mM hydrogen peroxide for 10 min served as positive control. PLA with single antibodies against Smad3 or PARP-1 are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b</b>) Quantification of the experiment shown in panel (a) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p

PLA of endogenous PARP-1 and PARP-2 ADP-ribosylation after TGFβ stimulation in HaCaT cells.

<p>(<b>a, c</b>) HaCaT cells were analyzed with PLA using antibodies against PARP-1 and PAR chains (<b>a</b>) or antibodies against PARP-2 and PAR (<b>c</b>) after stimulation with vehicle (0 min) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. PLA with single antibodies against PARP-1 or PAR are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b, d</b>) Quantification of the experiments shown in panels (a, c) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p

PARG regulates transcriptional responses to TGFβ.

<p>(<b>a–c</b>) Real-time RT-PCR analysis of endogenous <i>fibronectin (FN1)</i> (a), <i>PAI-1</i> (b) and control <i>PARP-1</i> (c) mRNAs in HaCaT cells transiently transfected with the indicated siRNAs (bottom of panel c) prior to stimulation (or not) with 5 ng/ml TGFβ1 for 9 h. The data are graphed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g007" target="_blank">Fig. 7g, h</a>. (<b>d–f</b>) Real-time RT-PCR analysis of endogenous <i>fibronectin (FN1)</i> (d), <i>PAI-1</i> (e) and control <i>PARG</i> (f) mRNAs in HaCaT cells transiently transfected with the indicated siRNAs (bottom of panel f) prior to stimulation (or not) with 5 ng/ml TGFβ1 for 9 h. The data are graphed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g007" target="_blank">Fig. 7g, h</a>. Stars (panels b–g) indicate statistical significance, <i>p</i><0.05. The figure shows representative experiments from four or more repeats.</p

PLA of endogenous Smad2/3 and PARP-2 complexes in HaCaT cells.

<p>(<b>a</b>) HaCaT cells were analyzed with PLA using antibodies against Smad2/3 and PARP-2 after transfection with control or the indicated specific siRNAs and stimulation with vehicle (-TGFβ) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. Cells stimulated with 10 mM hydrogen peroxide for 10 min served as positive control. PLA with single antibodies against Smad2/3 or PARP-2 are shown as controls. PLA images are shown as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1a</a>. (<b>b</b>) Quantification of the experiment shown in panel (a) following the histogram method of <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g001" target="_blank">Fig. 1b</a>. The figure shows a representative experiment from three or more repeats.</p

Regulation of Smad signaling by PARG and PARP-1.

<p>(<b>a, b</b>) Real-time RT-PCR analysis of endogenous <i>fibronectin (FN1)</i> (a) and <i>PAI-1</i> (b) mRNAs in HaCaT cells transiently transfected with the indicated siRNAs (bottom of panel b) prior to stimulation (or not) with 5 ng/ml TGFβ1 for 24 h. The data are graphed as in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone-0103651-g007" target="_blank">Fig. 7g, h</a>. Stars (panels a, b) indicate statistical significance, <i>p</i><0.05. The figure shows representative experiments from four or more repeats. (<b>c</b>) A model depicting TGFβ dimeric ligand that activates its cell surface type II (RII) and type I (RI) receptors, which phosphorylates Smad2 and Smad3, leading to oligomerization of Smad2, Smad3 and Smad4 into trimeric complexes. Smad oligomers enter the nucleus via nuclear pores and associate with chromatin in order to regulate transcription of target genes such as <i>Smad7</i>, <i>fibronectin (FN1)</i> and <i>PAI-1</i>. Nuclear PARP-1 and PARP-2 in complex associate with the Smad oligomer. For simplicity distinct complexes between Smads and PARP-1 and Smads and PARP-2 are not shown but their presence is supported by the experimental evidence. PARP-1/PARP-2 use NAD and oligo(ADP-ribosyl)ate Smad3 and Smad4 (ADP-ribose chains in red) and assist dissociation of Smads from DNA (as demonstrated in ref. <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone.0103651-Lnn2" target="_blank">[9]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone.0103651-Zhang1" target="_blank">[10]</a>, <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0103651#pone.0103651-Huang1" target="_blank">[11]</a>). PARG associates with ADP-ribose chains on the Smad complex and removes ADP-ribose units (ADPr) possibly generating mono(ADP-ribosyl)ated Smads (not shown). PARG therefore promotes Smad association with DNA and is required for optimal gene expression in response to TGFβ.</p

PLA of endogenous Smad3 ADP-ribosylation after TGFβ stimulation in HaCaT cells.

<p>(<b>a</b>) HaCaT cells were analyzed with PLA using antibodies against Smad3 and PAR chains after stimulation with vehicle (0 min) or with 2 ng/ml TGFβ1 for the indicated time periods. Specific RCA signals were detected in the nuclei. Cells stimulated with 10 mM hydrogen peroxide for 10 min served as positive control. PLA with single antibodies against Smad3 or PAR are shown as controls. PLA signals are shown in red, blue is DAPI staining for DNA and green is phalloidin staining for the actin cytoskeleton as a measure of overall cell architecture. (<b>b</b>) Quantification of the experiment shown in panel (a) using the DuolinkImageTool, with data plotted as a histogram divided in three classes according to the percent of cells that exhibit specific RCA signals: very low, 0–2 signals per cell [green]; low, 3–10 signals per cell [grey]; and high, >11 signals per cell [red]. The figure shows a representative experiment from three or more repeats.</p