Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Area of irradiated and unirradiated HeLa-Parp1 – CB-tagRFP cells. Cells on the left side of the dotted blue line were irradiated with 10 or 100 Cpp (orange and red target points, respectively) and allowed to recover for 1.5 h. For targeting, a single slice image of the Parp1 signal of the cells was taken (orange frame). Neighbouring cells on the right side of the blue dotted line were not irradiated and were used as controls. After 5EU incorporation and immunofluorescence staining, cells were imaged as z -stacks of 33 layers, with 300 nm distance in the single channels of Parp1, 5EU, γH2AX and phase contrast. Stacks were deconvolved for improved depth resolution. A single slice from the centre of the stack is shown. Arrows mark the target and hit areas verified by γH2AX. The best examples are the hit nucleoli in the three bottom cells (marked by asterisks). The outlines of these nucleoli in the phase contrast image still correlate with the outline before irradiation. The γH2AX foci are completely embedded in the nucleoli and at their sites; 5EU and Parp1 signals are less intense as in direct proximity. However, the irradiated nucleoli and the residual nucleoli of the corresponding nucleus do not show an overall decrease in 5EU signal. Fluctuations in brightness correlate with Parp1 fluctuations before irradiation and are also observable in control cells.

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: Irradiation, Immunofluorescence, Staining, Control

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Certain nucleoli hits after irradiation with 1, 10 and 50 Cpp and their influence on rRNA transcription. Recovery time was between 30 min and 7 h. Target definition images before irradiation are shown on the left. γH2AX, 5EU, Parp1 and phase contrast images of single cells were taken in the focus depth of the γH2AX foci after IF staining. Additionally, intensity line plots through the γH2AX foci along the white dotted line of the merged γH2AX+5EU image are shown on the right. Contrast was chosen such that 0.1% of the pixels were in saturation. The intensity plots show how 5EU and Parp1 signals (red and blue, respectively) were locally reduced at the damage site marked by γH2AX (black). Two-dimensional location is marked by yellow arrows. In these images, this correlates with local reduction of Parp1 and at higher doses additionally with altered appearance of the nucleolus in the phase contrast image. For better cell reconstruction, we used tiled images that consisted of 2×2 fields of view from the camera before irradiation and 3×3 after IF staining; thus, some cell images show the edge of the tiles. Scale bars: 3 µm.

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: Irradiation, Staining

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Cross-sections through z -stacks of certain nucleolus hit examples used for 5EU and Parp1 analysis. (A) 5EU and Parp1 signals were analysed in the central slice of the γH2AX foci (compare with Fig. 2 ). Nucleolar signal of 5EU is in green and γH2AX in red. The z -stacks shown consist of 13 slices with distance 300 nm, containing information of the single nucleus. (B) Analysis of single foci showing certain nucleolus hits on 5EU incorporation (left) and Parp1 distribution (right). Boxplots show median of the 5EU and Parp1 intensity ( I 5EU , I Parp1 ) ratios, in which the box represents the 2nd quartile, error bars show 1.5× the interquartile range and dots indicate outliers of the distribution. The signal intensity at the focus I …,focus minus the nucleus background signal I …,nucleus was calculated as a ratio of the signal intensity in the residual nucleolus I …,nucleolus minus the nucleus background signal I …,nucleus . 5EU and Parp1 signals were significantly reduced at the DNA damage site marked by γH2AX. A one-sample signed rank test (*) was used to test the hypothesis H 0 : median=1, which gave P <0.001. For the higher rates of 10 and 50 Cpp, 5EU incorporation and Parp1 intensity significantly decreased to about 0.2 and 0.5, respectively. The Mann–Whitney rank sum test was used to test the difference of the medians for 10 or 50 Cpp compared with 1 Cpp, which gave + P <0.001 for 5EU and ++ P <0.05 for Parp1. Scale bars: 3 µm (for all cross-sections).

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques: MANN-WHITNEY

Journal: Journal of Cell Science

Article Title: Local inhibition of rRNA transcription without nucleolar segregation after targeted ion irradiation of the nucleolus

doi: 10.1242/jcs.232181

Figure Lengend Snippet: Analysis of single foci showing certain nucleolus hits on 5EU incorporation and Parp1 distribution. Frequency of single foci, showing certain nucleolus hits with dips in the 5EU and Parp1 signals. If the ratio was below 0.80, the area was defined as a dip. Error bars describe the 95% confidence Wilson score intervals of the binomial proportion.

Article Snippet: A fluorescent, tagRFP-labelled chromobody (ChromoTek) marks the endogenous Parp1 protein in these cells.

Techniques:

Journal: Journal of medicinal chemistry

Article Title: Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

doi: 10.1021/acs.jmedchem.6b00990

Figure Lengend Snippet: Kinetic Parameters of PARP Full Length Enzymes and Catalytic Domain Fragments a

Article Snippet: Supernatants were analyzed by SDS-PAGE followed by Western Blotting using a mouse monoclonal HRP-conjugated anti-PARP1 antibody (Santa Cruz Biotechnology, sc-8007) for quantitation of endogenous full length PARP1 protein remaining in the soluble fraction.

Techniques: Construct

Journal: Journal of medicinal chemistry

Article Title: Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

doi: 10.1021/acs.jmedchem.6b00990

Figure Lengend Snippet: Enzymatic activities and inhibition of selected PARP enzymes and their isolated catalytic domains. (A) Nonlinear regression plots of the NAD+-dependent ADP-ribosylation rates of full length PARP1 and -2 and their catalytic domain fragments. (B) Domain arrangements of full length PARP1, -2, -3, and -10 proteins and the regions covered by the catalytic domain fragments. KM values (reported in detail in Table 1) are indicated by bars adjacent to each protein construct (black bars, full length proteins; gray bars, catalytic domain fragments). Domain designations: BRCT, BRCA1 carboxy terminal homology; PARP, ADP-ribosyltransferase domain; reg, regulatory subdomain; RRM, RNA recognition motif; SAM, sterile α motif domain; UIM, ubiquitin interacting motif; WGR, WGR-motif containing nucleic acid binding domain; ZnF, zinc finger domain. (C) Concentration response curves for in vitro inhibition of full length PARP1 and -2 and their catalytic fragments by olaparib. The IC50 value calculated from each data set is indicated. (D) Correlation of the IC50 values for olaparib, veliparib, rucaparib, and PJ34 determined using either full length enzymes or catalytic domain fragments of PARP1 (white), PARP2 (orange), and PARP10 (black). Full data are reported in Table 2 and Supporting Information, Figures S3−S8.

Article Snippet: Supernatants were analyzed by SDS-PAGE followed by Western Blotting using a mouse monoclonal HRP-conjugated anti-PARP1 antibody (Santa Cruz Biotechnology, sc-8007) for quantitation of endogenous full length PARP1 protein remaining in the soluble fraction.

Techniques: Inhibition, Isolation, Construct, Sterility, Ubiquitin Proteomics, Binding Assay, Concentration Assay, In Vitro

Journal: Journal of medicinal chemistry

Article Title: Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

doi: 10.1021/acs.jmedchem.6b00990

Figure Lengend Snippet: In Vitro Potencies of PARP Inhibitors a

Article Snippet: Supernatants were analyzed by SDS-PAGE followed by Western Blotting using a mouse monoclonal HRP-conjugated anti-PARP1 antibody (Santa Cruz Biotechnology, sc-8007) for quantitation of endogenous full length PARP1 protein remaining in the soluble fraction.

Techniques: In Vitro, Construct

Journal: Journal of medicinal chemistry

Article Title: Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

doi: 10.1021/acs.jmedchem.6b00990

Figure Lengend Snippet: Selective vs broad PARP inhibition and their structural basis. Concentration−response curves for PARP inhibitor dependent in vitro inhibition of full length PARP1,-2, -3, and-10 and the catalytic fragments of tankyrase-1 and -2. For clarity, plots are shown only for a selection of PARP family members; full details and IC50 values calculated from these data are reported in Table 2 and Supporting Information, Figures S3−S8. Symbol colors are explained in the legends and pertain to all panels. Homologous key side chains are shown in all structure panels to facilitate orientation. (A) Concentration−response curves for veliparib. (right panel) Crystal structures of veliparib bound to PARP1 (in pink; PDB 2RD6) and PARP2 (in green),37 with key side chain interactions indicated. A water mediated interaction with PARP2-E335 in the α-helical regulatory subdomain is conserved in PARP1 (involving D766) but not in any other PARP family member. A structure of PARP10 with veliparib shows the ligand bound in a similar orientation albeit without making interactions with the protein outside the nicotinamide pocket (Supporting Information, Figure S13). (B) Concentration−response curves for niraparib. (right panel) Crystal structure of niraparib bound to PARP1. Niraparib selectivity for PARP1 and -2 is rationalized by interactions with the regulatory subdomain via a hydrogen bond to D766 as for veliparib. (C) Concentration−response curves for olaparib. (right panel) Crystal structure of olaparib bound to PARP2. Olaparib forms several hydrogen bonds with backbone atoms in the catalytic domain including R444 and water mediated hydrogen bond with the D339 and van der Waals interactions with the aliphatic part of the E335 side chain in the regulatory domain. (D) Concentration−response curves for talazoparib. (right panel) Crystal structure of talazoparib bound to PARP1. Talazoparib efficiently fills the nicotinamide and N-ribose subpockets, forming a water mediated hydrogen bond with the catalytic residue E988 and van der Waals interactions on both sides of the cleft. (E) Concentration−response curves for rucaparib. (right panel) Crystal structure of rucaparib bound to PARP1 (pink) and TNKS2 (orange; PDB 4BJC). (F) Concentration−response curves for PJ34. (right panel) Crystal structure of PJ34 bound to PARP1 (pink) showing two conformations of the inhibitor’s dimethyl glycinamide moiety. PJ34 binding to PARP3 (cyan; PDB 3CE0), PARP15 (gray; PDB 3GEY), TNKS1 (yellow; PDB 3UH2), and TNKS2 (blue; PDB 4BJB) utilizes similar interactions in the nicotinamide subpocket but takes advantage of nonpolar features in the pocket by yet different conformations of the dimethyl glycinamide moiety.

Article Snippet: Supernatants were analyzed by SDS-PAGE followed by Western Blotting using a mouse monoclonal HRP-conjugated anti-PARP1 antibody (Santa Cruz Biotechnology, sc-8007) for quantitation of endogenous full length PARP1 protein remaining in the soluble fraction.

Techniques: Inhibition, Concentration Assay, In Vitro, Selection, Residue, Binding Assay

Journal: Journal of medicinal chemistry

Article Title: Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

doi: 10.1021/acs.jmedchem.6b00990

Figure Lengend Snippet: The potent tankyrase inhibitor XAV939 inhibits PARP1 with submicromolar potency in vitro and in cells. (A) Potencies of tankyrase inhibitors. Schematic representation of PARP inhibitor potencies mapped on a phylogenetic tree of human PARP enzymes. Data for IWR-1 inhibition of tankyrase-1 and all data for 3 were taken from previous publications.16,17 Experimental data underlying the remainder are presented in Table 3 and Supporting Information, Figures S10−S12. Red sphere sizes are proportional, on a logarithmic scale, to IC50 values in the range 1 nM to 10 μM; red dots indicate IC50 values higher than 10 μM; black dots indicate no inhibition detected. (B) Concentration−response curves for in vitro inhibition of selected PARP and tankyrase enzymes by XAV939 and IWR-1. (C) (left panel) Structural alignment of tankyrase-2 (gold; PDB 3KR8)47 and PARP1 (pink; reported here), both in complex with XAV939. Tankyrase-2 side chains that contribute to XAV939 interactions are shown. In the PARP1 α-helical regulatory domain (which is missing in the tankyrases), aliphatic side chains interacting with the trifluoromethyl group of XAV939 are shown. (right panel) Structural alignment of the tankyrase-2 IWR-1 complex70 (gold) with PARP1−XAV939 (pink). IWR-1 does not engage in interactions with the nicotinamide pocket but is bound in the adenine subsite. (D) Concentration−response fingerprints of PARP1 in HEK293 cells following preincubation with either XAV939 or olaparib. Both compounds engage PARP1 in cells. (E) Inhibition of PARP1 activity in H2O2-treated HEK293 cells preincubated with either XAV939 or PJ34.

Article Snippet: Supernatants were analyzed by SDS-PAGE followed by Western Blotting using a mouse monoclonal HRP-conjugated anti-PARP1 antibody (Santa Cruz Biotechnology, sc-8007) for quantitation of endogenous full length PARP1 protein remaining in the soluble fraction.

Techniques: In Vitro, Inhibition, Concentration Assay, Activity Assay

Journal: Journal of medicinal chemistry

Article Title: Structural Basis for Potency and Promiscuity in Poly(ADP-ribose) Polymerase (PARP) and Tankyrase Inhibitors

doi: 10.1021/acs.jmedchem.6b00990

Figure Lengend Snippet: In Vitro Potencies of Tankyrase Inhibitors a

Article Snippet: Supernatants were analyzed by SDS-PAGE followed by Western Blotting using a mouse monoclonal HRP-conjugated anti-PARP1 antibody (Santa Cruz Biotechnology, sc-8007) for quantitation of endogenous full length PARP1 protein remaining in the soluble fraction.

Techniques: In Vitro, Construct

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Co-immunoprecipitation of endogenous SET8 and PARP1 in HCT116 cells. Rabbit IgG (left lane) was used as a negative control. (B) Colocalization between FLAG-PARP1 (red), GFP-SET8 (green) and endogenous PCNA (pink) in COS-7 cells. DAPI (blue) represents the nuclear DNA content. (C) Mapping of domain interactions using GST-pulldown assays between SET8 domains (top, left) and full-length recombinant PARP1 protein and GST-pulldown assays between PARP1 domains and full-length recombinant SET8 protein (top, right). The PARP1 or SET8 binding were detected by western blotting using PARP1 antibody (middle, left) or SET8 antibody (middle, right) respectively. Ponceau stain gels (bottom) represent the amount of GST beads constructs used for the GST-pulldown assays. (D) In vitro detection of full-length recombinant SET8 ADP-ribosylation by full-length recombinant PARP1 by western blotting using anti-ADP ribose antibody (top). Ponceau stain gels (bottom) represent the amount of PARP1 and SET8 recombinant enzyme used for ADP-ribosylation assay (bottom). (E) Detection of SET8 lysines ADP-ribosylation using mass spectrometry analysis of SET8 ADP-ribosylated peptides by full-length recombinant PARP1 protein in vitro .

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Immunoprecipitation, Negative Control, Recombinant, Binding Assay, Western Blot, Staining, Construct, In Vitro, Mass Spectrometry

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Detection of unbound 100 bp DNA ladder by TBE ethidium bromide-stained gel in supernatants (left lane) on GST-SET8 domains or mutant (M) using GST-pulldown assays (top, left side). Asterisks are representing shift of DNA on GST-SET8 157-352 amino acid protein or GST-SET8 full-length protein (FL) beads. Ponceau stain represents the amount of GST beads constructs used for the GST-pulldown (bottom, left side). (B) Different concentrations of recombinant full-length SET8 protein binding to DNA using EMSA to determine the equilibrium dissociation constant (Kd). (C) Detection of unbound mononucleosome by TBE ethidium bromide-stained gel in supernatants on GST beads versus GST-SET8 domains or mutant (M) beads using GST-pulldown assays. (D) Detection of unbound DNA (left side) or unbound mononucleosome (right side) by TBE ethidium bromide-stained gel in supernatants (top) on GST beads versus GST-SET8 FL beads using GST-pulldown assays. GST or GST-SET8 FL beads were poly ADP-ribosylated or not (with or without NAD) using full-length recombinant PARP1 as demonstrated by western blot using anti-ADP ribose antibody (middle). Ponceau stain gel (bottom) represents the amount of GST bead constructs used for the GST-pulldown and the ADP-ribosylation western blot analysis. (E) SET8 histone methyltransferase assay on full-length recombinant histone H4 using full-length recombinant SET8 in presence or absence of activated full-length recombinant PARP1.

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Staining, Mutagenesis, Construct, Recombinant, Protein Binding, Western Blot, HMT Assay

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) GFP-SET8 immunoprecipitation in overexpressed GFP-SET8 COS-7 cells with or without FLAG-PARP1 overexpression in presence or not of the proteasome inhibitor MG132. Western blots detecting the amount of GFP-SET8 protein overexpressed in total extract (top, left) as well as the amount of FLAG-PARP1 protein overexpressed (top, right) using anti-GFP and anti-FLAG antibody respectively. Western blots detecting the amount of ubiquitin (Ub) (bottom, left) or ADP-ribosylation (bottom, right) of immunoprecipitated GFP-SET8 protein using anti-HA and anti-ADP ribose antibody respectively. Anti-actin was used as a loading control (middle). (B) GFP-SET8 immunoprecipitation in overexpressed GFP-SET8 COS-7 cells in presence or not (DMSO) of Cullin inhibitor (Cul4Ai) or PARG inhibitor (PARGi). Western blot detection of total GFP-SET8 protein levels overexpressed in COS-7 cells (top). Anti-actin antibody was used as control (middle). Western blot detecting the amount of poly-ADP ribosylation (bottom) of immunoprecipitated GFP-SET8 protein using an anti-ADP ribose antibody. (C) Western blots (left side) detecting the amount of PARP1 (top), SET8 protein (middle) as well as the amount of H4K20me1, H4K20me2 and H4K20me3 levels (bottom) in total protein extract of knockdown HeLa cells treated with esiRNA GFP (control), esiRNA PARP1 and esiRNA SET8, respectively. Respective densitometry analyses of protein abundance representative of at least 2 biological experiments are shown (right side). Ponceau stain was used as control (left side).

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Immunoprecipitation, Over Expression, Western Blot, Ubiquitin Proteomics, Control, Knockdown, esiRNA, Quantitative Proteomics, Staining

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Western blot indicating PARP1 (top, left), SET8 (middle, left) and H4K20me1 (middle, left) levels in total protein extracts from HeLa cells synchronized in G1, S and G2/M phases, respectively. Western blot of CDT1 protein levels is shown as a cell cycle synchronization control as well as Ponceau stain for loading control and densitometry analyses (bottom, left). Respective densitometry analyses of H4K20me1 (top, right) and SET8 (bottom, right) relative protein abundances are shown (right) and representative of at least 2 biological experiments. (B) SET8 immunoprecipitation from total protein extract in HeLa cells synchronized in G1, S and G2/M phases. Western blots detection of PARP1 (top, left) as well as SET8 immunoprecipitated protein levels (bottom, left) are revealed. Densitometry analyses of PARP1/SET8 ratio during G1, S and G2/M cell cycle phases are shown (right) and are representative of at least 2 biological experiments. (C) Pulsed chased cells with 5-ethynyl-2 -deoxyuridine (EdU) to label DNA (magenta) is transfected with GFP-SET8 (green). Endogenous ADP-ribose (red) is revealed by anti-ADP-ribose conjugated with Texas Red. Merged images demonstrates the co-localization of EDU, SET8 and ADP-ribose.

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Western Blot, Control, Staining, Immunoprecipitation, Transfection

Journal: bioRxiv

Article Title: Poly ADP-ribosylation of SET8 leads to aberrant H4K20 methylation domains in mammalian cells

doi: 10.1101/2021.11.13.468478

Figure Lengend Snippet: (A) Genome-wide metagene plot showing H4K20me1 profile (ChIP-seq) in control and PARP1 knockdown HeLa cells. (B) Genome-wide metagene plot showing H4K20me3 profile (ChIP-seq) in control and PARP1 knockdown HeLa cells. (C) Pearson correlation of H4K20me1 and H4K20me3 regions in PARP1 knockdown HeLa cells and its control. (D) Peak width profile of H4K20me1 by binning the peaks into different lengths in PARP1 knockdown cells and its control. (E) Peak width profile of H4K20me3 by binning the peaks into different lengths in PARP1 knockdown cells and its control (F) Spearman correlation of H4K20me1 regions in the human (hg38) genes and open chromatin region in PARP1 knockdown cells and its control. (G) Spearman correlation of H4K20me3 regions in the human (hg38) genes and open chromatin region in PARP1 knockdown cells and its control (H) Representative IGV genomic tracks showing H4K20me1 and H4K20me3 profile in PARP1 knockdown cells and its control.

Article Snippet: Co-immunoprecipitation of endogenous PARP1 and SET8 were performed with 200 μg of total extract from crosslinked (1% formaldehyde for 10 min) HCT116 or HeLa cells using anti-PARP1 antibody (Cell Signaling Technology # 9532), anti-SET8 antibody (Santa Cruz Biotechnology # sc-515433) or 5 μg of rabbit IgG as a control antibody (Santa Cruz Biotechnology # sc-2027).

Techniques: Genome Wide, ChIP-sequencing, Control, Knockdown

Journal: Oncogene

Article Title: Poly(ADP-ribose)-dependent regulation of Snail1 protein stability.

doi: 10.1038/onc.2011.153

Figure Lengend Snippet: Figure 1 Snail1 downregulation and E-cadherin accumulation induced by PARP inhibition and PARP-1 knockdown. (a) Treatment with the PARP inhibitor PJ-34 (10 mM) or KU0058948 (100 mM) for 22 h (PARP-1 inhibitors) reduces Snail1 or Vimentin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) levels and increase the E-cadherin levels (Santa Cruz Biotechnology) in A375 human melanoma cells. (b) Knockdown of PARP-1 by siRNA in A375 human melanoma cells induces downregulation of Snail1 and subsequent increase in the expression of E-cadherin. (c and d) Treatment with the PARP inhibitor PJ-34 or KU0058948 (left panel), or knockdown of PARP-1 by siRNA (right panel) produces the same effect in G361 human melanoma cells than in A375 cells. Both cells were transiently transfected with an irrelevant siRNA (SIMA) or PARP-1 siRNA for 24h using Lipofectamine Plus Reagent (Invitrogen, Carlsbad, CA, USA) according to the manufacturer’s recommendations. At 48 h post-transfection, the expression of E-cadherin and Snail1 was measured. Cells were washed twice in phosphate-buffered saline (PBS) and scraped in Laemmli buffer (1 M Tris, 20% SDS and 10% glycerol) and sonicated. The protein concentration was determined using the Lowry assay. Levels of b-actin were monitored as a loading control. Immunoreactive bands were visualized with the ECL Plus system (Amersham Biosciences, Piscataway, NJ, USA). The G361 cells were cultured in Dulbecco’s modified Eagle’s medium (DMEM) supplemented with 10% inactivated fetal bovine serum (FBS) and gentamicin (Gibco, Carlsbad, CA, USA). The A375 cells were given by Dr Bosserhoff (Institute of Pathology, University Regensburg, Germany). Cells were maintained in DMEM supplemented with penicillin (50 U/ml), streptomycin (50 mg/ml),

Article Snippet: The inhibition of PARP using [N-(6-Oxo-5,6-dihydro-phenanthridin-2-yl)N,N-dimethylacetamide] (PJ-34) or KU0058948 in the human melanoma cell lines A375 or G361 downregulated endogenous protein Snail1 as well as mRNA levels and Snail1dependent transcriptional activation (Figures 1a (A375) and 1c (G361) and Supplementary Figures S1a and b C on tro l PJ -3 4 Snail1 PARP-1 Snail1 E-cadherin SI M A iP AR P1 PARP-1 Snail1 E-cadherin KU 00 58 94 8 Vimentin E-cadherin Snail1 Vimentin E-cadherin -actin -actin -actin -actin -actin 1 1 1 1 11 1 1 1 1 1 1 SI M A iP AR P1 C on tro l PJ -3 4 KU 00 58 94 8 2.823.62 0.520.24 0.14 0.07 3.73 1.582.56 0.060.16 0.660.58 1.26 0.26 0.32 0.280.45 Figure 1 Snail1 downregulation and E-cadherin accumulation induced by PARP inhibition and PARP-1 knockdown. (a) Treatment with the PARP inhibitor PJ-34 (10mM) or KU0058948 (100mM) for 22 h (PARP-1 inhibitors) reduces Snail1 or Vimentin (Santa Cruz Biotechnology, Santa Cruz, CA, USA) levels and increase the E-cadherin levels (Santa Cruz Biotechnology) in A375 human melanoma cells. (b) Knockdown of PARP-1 by siRNA in A375 human melanoma cells induces downregulation of Snail1 and subsequent increase in the expression of E-cadherin. (c and d) Treatment with the PARP inhibitor PJ-34 or KU0058948 (left panel), or knockdown of PARP-1 by siRNA (right panel) produces the same effect in G361 human melanoma cells than in A375 cells.

Techniques: Inhibition, Knockdown, Expressing, Transfection, Saline, Sonication, Protein Concentration, Lowry Assay, Control, Cell Culture