(A) cDNA microarray analysis of mRNA in Smad4-deficient MDA-MB-468 cells after infection with adenovirus expressing LacZ or Smad4 and stimulation with 2 ng/ml TGFβ1 or 300 ng/ml BMP7

Normalized mean expression values with error bars from triplicate microarray expressions are shown (arbitrary units). (B and C) Semiquantitative RT-PCR analysis of and mRNA in MDA-MB-468 cells as in A (B) or in response to 2 ng/ml TGFβ1 in HaCaT cells (C). Amplified cDNA sizes are in bp. (D) SIK and α-tubulin loading control protein profiles in response to 5 ng/ml TGFβ1 in HaCaT cells. Protein sizes are in kilodaltons. (E) Induction of nuclear and cytoplasmic levels of endogenous SIK by 5 ng/ml TGFβ1 in HaCaT cells (bar, 10 μm).<p><b>Copyright information:</b></p><p>Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(4):655-662.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518705.</p><p></p

(A) Immunoblot of endogenous SIK and α-tubulin loading control in HaCaT TCL after transfection with control (siLuc) and specific (siSIK) siRNAs and 12 h of stimulation with 5 ng/ml TGFβ1

The asterisk marks a nonspecific protein band. (B) Quantitative real-time RT-PCR analysis in HaCaT cells treated with siRNAs as in A and stimulated with 5 ng/ml TGFβ1 for 6 h ( and ) or 8 h ( and ). Mean fold induction of TGFβ-stimulated mRNA levels (black bars) relative to unstimulated levels (gray bars) are plotted, with standard errors determined from triplicate or quadruplicate samples. Asterisks indicate significant difference ( test: P <p><b>Copyright information:</b></p><p>Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(4):655-662.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518705.</p><p></p

(A) Coimmunoprecipitation of CA-ALK5 with wild-type (WT) SIK and TCL controls for SIK

Low levels of SIK coexpressed with high levels of CA-ALK5 allowed detection of SIK–ALK5 complexes with only small effects on receptor down-regulation. (B) Coimmunoprecipitation of wild-type SIK with wild type and various Smad7 domains (C, C-terminal; Link, linker; N, N-terminal). TCL controls for SIK and immunoglobulin chains (Ig) are shown. (C) Coimmunoprecipitation of endogenous SIK with endogenous ALK5 and Smad7. HaCaT cell lysates, pretreated with 50 μM MG132 and 5 ng/ml TGFβ1 overnight, were immunoprecipitated with anti-ALK5, anti-Smad7, and anti-SIK (988) antibodies, followed by immunoblotting with anti-SIK (988) antibody. TCL immunoblots with the three antibodies demonstrate specificity of each antiserum. Asterisks indicate specific protein bands and sizes are in kilodaltons. The top left panel (Mock and ALK5) was exposed four times longer than the top right panel (Smad7 and SIK). (D) Triple confocal immunofluorescence analysis of wild-type SIK, CA-ALK5, and wild-type Smad7 in transfected Mv1Lu cells. Insets show high magnifications of peripheral clusters just below the plasma membrane (bar, 10 μm). (E) SIK represses synergistically with Smad7 CAGA promoter induction by 0.5 ng/ml TGFβ1 for 6 h in HepG2 cells transfected with empty vector or Flag-SIK and Flag-Smad7. Normalized promoter activity is plotted as mean values from triplicate determinations with standard error bars. (F) Depletion of endogenous Smad7 stabilizes the CA-ALK5 receptor and wild-type SIK. Immunoblot of HEK293T cell extracts after transient transfection with shRNA vector pSuper-Smad7 or its empty (mock) version together with increasing amounts (triangles) of GFP-SIK and constant amount of CA-ALK5. The asterisks indicate nonspecific protein bands. Protein sizes are shown in kilodaltons.<p><b>Copyright information:</b></p><p>Taken from "TGFβ induces SIK to negatively regulate type I receptor kinase signaling"</p><p></p><p>The Journal of Cell Biology 2008;182(4):655-662.</p><p>Published online 25 Aug 2008</p><p>PMCID:PMC2518705.</p><p></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 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

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

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

Over-expression of glycanated serglycin increases breast cancer cell migration.

<p>Cells (5×10⁵) were plated in triplicates in 12 well plates and cultured until confluence. Then wounds were made using a sterile pipette tip, debris was removed and fresh culture medium was added. The cells were monitored at 0, 24 and 48 h and were photographed (A). Wound areas were quantified at various time intervals using Image J software (B). Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (<i>p</i><0.05). (C) Migratory properties of the cells were also evaluated by Transwell migration assay. 1×10⁵ cells were suspended in culture medium supplemented with 0.5% FBS and loaded onto the top of Transwell chambers. Cells were then maintained in Transwell chambers for 48 h with 10% FBS as chemotactic stimuli in the bottom chamber. Transmigrating cells were stained with Giemsa and counted. Data are given as means and SD of three independent experiments. Statistical significance of variances was calculated using a one-way ANOVA test. Asterisk (*) indicates statistically significant differences (<i>p</i><0.05). (D) In a set of experiments, cells were cultured on glass coverslips. After wounding with a sterile pipette tip, debris was removed, and cells were either immediately fixed in 3% formaldehyde in PBS (t = 0 h) or cultured for 24 h and then fixed. Immunofluorescence staining for serglycin (green), nuclei (blue) and F-actin (red) in MCF-7VSG cells was performed. Bars, 25 µm.</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