Journal: bioRxiv

Article Title: A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

doi: 10.1101/707661

Figure Lengend Snippet: (A) Schemes of TOP1-His-IP pull-down, TOP2α- and TOP2β-FLAG-IP pull-down for LC-MS/MS. (B) Scheme of PIAS4 FLAG-IP pull-down for LC-MS/MS. (C) HEK293 cells were transfected with indicated siRNAs, followed by treatment with 20 μM CPT for 30 min (same conditions used for the following DUST assays unless otherwise indicated). Representative DUST assay for immunodetection of SUMO-2/3-, SUMO-1-, Ub- and total TOP1-DPCs. (D) HEK293 cells were transfected with control, RNF4 or RNF111 siRNA, followed by CPT treatment and DUST assay for detection of SUMO-, Ub- and total TOP1-DPCs. (E) HEK293 cells were transfected with indicated siRNAs followed by treatment with 200 μM ETP for 30 min (same conditions used for the following DUST assays unless otherwise indicated). DUST assay for immunodetection of SUMO-2/3-, SUMO-1-, Ub- and total TOP2-DPCs.

Article Snippet: Recombinant human PIAS4 was purchased from OriGene (Cat. # TP306748).

Techniques: Liquid Chromatography with Mass Spectroscopy, Transfection, Immunodetection

Journal: bioRxiv

Article Title: A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

doi: 10.1101/707661

Figure Lengend Snippet: (A) Cells were transfected with the 6×His tagged TOP1 expression splasmid, followed by treatments with DMSO or 20 μM CPT in absence or presence of 10 μM MG132 for 30 min. Native His-pull down was performed as described under “Materials and Methods.” His-pull down samples and cell lysates (input) were then subjected to IB using indicated antibodies. (B) Cells were transfected with the FLAG tagged TOP2α expression plasmid, followed by treatments with DMSO or 200 μM ETP in absence or presence of MG132 for 30 min. Native FLAG-IP was performed as described under “Materials and Methods.” IP samples and cell lysates (input) were then subjected to IB using indicated antibodies. (C) Cells were transfected with the FLAG tagged TOP2β expression plasmid, followed by treatments with DMSO or ETP in the absence or presence of MG132 for 30 min. IP samples and cell lysates were subjected to IB using indicated antibodies. (D) U2OS cells were transfected with empty vector (EV), FLAG-PIAS4 or RNF4-FLAG expression plasmids then subjected to DMSO or CPT treatment (20 μM, 30 min), followed by proximity ligation assay using anti-TOP1 antibody and anti-FLAG antibody. Scale bar represents 10 microns. (E) U2OS cells were transfected with FLAG-PIAS4 or RNF4-FLAG expression plasmids then subjected to DMSO or ETP treatment (200 μM, 30 min), followed by PLA using anti-TOP2α antibody and anti-FLAG antibody. Scale bar represents 10 microns. (F) Cells were transfected with indicated siRNAs, followed by WB of the whole cellular lysates to validate downregulation of each protein.

Article Snippet: Recombinant human PIAS4 was purchased from OriGene (Cat. # TP306748).

Techniques: Transfection, Expressing, Plasmid Preparation, Proximity Ligation Assay

Journal: bioRxiv

Article Title: A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

doi: 10.1101/707661

Figure Lengend Snippet: (A) Left panel: SUMO conjugation assay with recombinant TOP1 incubated with SUMO E1, SUMO E2, SUMO-1, and increasing concentration of PIAS4. Reaction products were separated by SDS-PAGE and monitored by IB using anti-SUMO-1 antibody. Right panel: same as left panel except the IB was performed with anti-SUMO-2/3 antibody. (B)&(C) same as panel (A) except TOP2α and TOP2β were used instead of TOP1, respectively. (D) Ubiquitin conjugation assay with recombinant TOP1 and RNF4. Unmodified TOP1 (left) or in vitro SUMOylated TOP1 in the presence of SUMO E2 and PIAS4 was tested for ubiquitin conjugation in the presence of Ub E1, Ub E2 and the indicated concentrations of RNF4. Reaction products were separated by SDS-PAGE and monitored by IB using anti-Ub antibody. (E)&(F) same as panel (D) except TOP2α and TOP2β were used instead of TOP1, respectively.

Article Snippet: Recombinant human PIAS4 was purchased from OriGene (Cat. # TP306748).

Techniques: Conjugation Assay, Recombinant, Incubation, Concentration Assay, SDS Page, In Vitro

Journal: bioRxiv

Article Title: A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

doi: 10.1101/707661

Figure Lengend Snippet: (A) HCT116 WT and PIAS4-KO cells transfected with indicated plasmids were treated with CPT and analyzed by DUST assay for detection of SUMO-2/3-, SUMO-1- and Ub-TOP1-DPCs. (B) Same as panel (A) except that cells were treated with ETP to induce TOP2-DPCs. (C) Left, Scheme of human PIAS4. Right, Scheme of human RNF4. (D) MCF7 WT and RNF4 KO cells were transfected with indicated plasmids, followed by CPT treatment and analyzed by DUST assay for detection of SUMO-2/3-, SUMO-1- and Ub-TOP1-DPCs. (E) Same as panel (D) except that cells were treated with ETP to induce TOP2-DPCs. (F) Upper panel, MCF7 RNF4-KO cells were transfected with RN4 WT or H156A plasmids, followed by CPT treatment (20 μM, 1 h) and DUST assay for immunodetection of Ub-TOP1-DPCs. Lower panel: Same as upper panel except that ETP (200 μM, 1 h) was used to induce TOP2-DPCs.

Article Snippet: Recombinant human PIAS4 was purchased from OriGene (Cat. # TP306748).

Techniques: Transfection, Immunodetection

Journal: bioRxiv

Article Title: A conserved SUMO-Ubiquitin pathway directed by RNF4/SLX5-SLX8 and PIAS4/SIZ1 drives proteasomal degradation of topoisomerase DNA-protein crosslinks

doi: 10.1101/707661

Figure Lengend Snippet: (A) WB of HCT116 WT and PIAS4 knockout (KO) cells using anti-PIAS4 antibody and anti-GAPDH antibody. (B) WB of MCF7 WT and RNF4 KO cells with or without RNF4-FLAG overexpression (OE) using anti-RNF4 antibody and anti-tubulin antibody. (C) HCT116 cells were transfected with empty vector (EV), FLAG-PIAS4 WT, FLAG-PIAS4, FLAG-PIAS4 SAPΔ or FLAG-PIAS4 C342A expression plasmid for 48 hours, WB using anti-FLAG antibody and anti-GAPDH antibody. (D) MCF7 cells were transfected with empty vector (EV) or RNF4-FLAG WT, RNF4-FLAG SIMΔ or RNF4-FLAG H156A expression plasmid for 48 hours, followed by WB using anti-FLAG antibody and anti-GAPDH antibody. (E) Left panel: U2OS cells were transfected with the indicated plasmids (FLAG-tagged) before CPT treatment (20 μM, 30 min), followed by PLA using anti-TOP1 and anti-FLAG antibodies. Right panel: U2OS cells were transfected with the indicated plasmids (FLAG-tagged) before ETP treatment (200 μM, 30 min), followed by PLA using anti-TOP2α and anti-FLAG antibodies. Scale bar represents 10 microns. (F) Quantitation of foci per cells of each treatment group as shown in panel (E).

Article Snippet: Recombinant human PIAS4 was purchased from OriGene (Cat. # TP306748).

Techniques: Knock-Out, Over Expression, Transfection, Plasmid Preparation, Expressing, Quantitation Assay

Journal: Journal of cellular and molecular medicine

Article Title: Aryl Hydrocarbon Receptor Alleviates Hepatic Fibrosis by Inducing Hepatic Stellate Cell Ferroptosis.

doi: 10.1111/jcmm.70278

Figure Lengend Snippet: FIGURE 1 | AHR directly regulates Mrp1 transcription in mHSCs. (A–C) The expression of Ahr, Mrp1 or Cyp1a1 was detected by QPCR. (D) There are two potential AHR exogenous response elements on the Mrp1 promoter sequence. (E) Promoter sequences containing XREL1 and XREL2 were cloned onto PGL3 plasmids, and mutant plasmids were constructed. (F) Double luciferase assay to detect luciferase activity. (G) EMSA detects AHR binding to specific elements XREL1 and XREL2. (H) CHIP detects AHR binding to specific elements XREL1 and XREL2. Data are expressed as means ± SD; *p < 0.05, **p < 0.01 and ***p < 0.001; Student's t-test or one-way ANOVA.

Article Snippet: ChIP assays were performed using the ChIP Assay kit (Beyotime, P2078). mHSCs were treated with YH439 (5 μM) for 24 h. Subsequently, mHSCs were sonicated and then immunoprecipitated with the antibody against AHR (1:100 dilution BOSTER Cat# A00225- 4, RRID: AB_3095576) with IgG (1:100 dilution Proteintech Cat# 30000- 0- AP RRID AB_2819035) as a negative control.

Techniques: Expressing, Sequencing, Clone Assay, Mutagenesis, Construct, Luciferase, Activity Assay, Binding Assay

Journal: Proceedings of the National Academy of Sciences of the United States of America

Article Title: The serine/arginine-rich protein SF2/ASF regulates protein sumoylation

doi: 10.1073/pnas.1004653107

Figure Lengend Snippet: SF2/ASF promotes the transfer of SUMO from Ubc9 to p53. (A) SF2/ASF does not stimulate Ubc9-SUMO1 thioester bond formation. Recombinant E1 (150 ng), E2 (100 ng), and SUMO1 (200 ng) were incubated as described in Material and Methods for the indicated time points, either with or without GST-SF2/ASF (200 ng). Reactions were stopped by addition of an equal volume of 2× nonreducing SDS-sample buffer (lanes 1–6) or 2× Laemmli sample buffer containing 100 mM DTT (lane 7), and analyzed by Western blotting with an anti-Ubc9 antibody. (B) SF2/ASF stimulates SUMO1 transfer from Ubc9 to p53. Recombinant E1 (150 ng), E2 (300 ng), and SUMO1 (200 ng) were incubated as described in Materials and Methods. After diluting the reaction in EDTA-containing buffer, the SUMO1-loaded E2 was incubated with GST-p53 (200 ng), either with or without GST-SF2/ASF (200 ng) for the indicated time points. In lanes 7 and 8, EDTA was omitted, enabling further rounds of Ubc9 loading and transfer (“multiple turnover”). Reactions were stopped by addition of an equal volume of 2× Laemmli sample buffer and analyzed by SDS/PAGE, followed by Western blotting with an anti-p53 antibody.

Article Snippet: SUMO E1, SUMO E2, GST-p53, GST-RanBP2ΔFG, and GST-Sp100 were from BIOMOL/Enzo Life Sciences.

Techniques: Recombinant, Incubation, Western Blot, SDS Page

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Sequences of oligonucleotides used in experiments

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Sequencing

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Effect of uracil DNA glycosylase activity on oligonucleotide substrates containing a single U:A base pair or U:G mismatch. Incubation with recombinant Escherichia coli uracil DNA glycosylase led to enzyme concentration dependent ( A ) and time-dependent ( B ) decreases in the amount of intact U:A (open symbols) and U:G (closed symbols) containing substrate retained in the wells after alkaline denaturation. For the time course analysis squares, circles and triangles indicate data generated with 0.1, 0.2 or 0.4 units/well, respectively, for the U:A containing substrate or with 0.0125, 0.025 or 0.05 units/well, respectively, for the U:G containing substrate. Incubation with HepG2 nuclear extract ( C ) or peripheral blood mononuclear cell pooled extract ( D ) also caused concentration-dependent decreases in the amount of intact U:A and U:G containing substrate retained in the wells. For both substrates, the apparent uracil DNA glycosylase activity was directly proportional to the concentration of HepG2 ( E ) and peripheral blood mononuclear cell ( F ) nuclear extract over the entire range tested ( r 2 = 0.99 in both cases). Data shown represent the mean ± SD of triplicate technical replicates except for time course data for which duplicates were analyzed.

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay, Incubation, Recombinant, Concentration Assay, Generated

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Assay for DNA polymerase repair activity. Incubation of an immobilized hairpin loop oligonucleotide substrate containing a single nucleotide gap on one strand within the double-stranded region with exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I, in the presence of dNTPs, ATP and excess T4 DNA ligase, led to an increase in intact substrate retained in the microplate wells following neutral denaturation that was dependent on the concentration of the polymerase ( A ). Incubation with HepG2 nuclear extract ( B ) or peripheral blood mononuclear cell pooled nuclear extract ( C ) also caused concentration-dependent increases in the amount of intact substrate retained in the wells. The apparent DNA polymerase activity was directly proportional to the concentration of nuclear extract over the range 0–0.06 μg/well for the HepG2 nuclear extract ( r 2 = 0.98) ( D ) and over the range 0–0.25 μg/well for the peripheral blood mononuclear cell nuclear extract ( r 2 = 0.99) ( E ). Data shown represent the mean ± SD of triplicate technical replicates.

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay, Incubation, Concentration Assay

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Assay for DNA ligase activity. Incubation of an immobilized hairpin loop oligonucleotide substrate containing a single ligatable nick in one strand within the double-stranded region with T4 DNA ligase, in the presence of ATP, led to an increase in intact substrate retained in the microplate wells following neutral denaturation that was dependent on the concentration of the polymerase ( A ). Incubation with HepG2 nuclear extract ( B ) and peripheral blood mononuclear cell pooled nuclear extract ( C ) also caused concentration-dependent increases in the amount of intact substrate retained in the wells. The apparent DNA ligase activity was directly proportional to the concentration of HepG2 ( D ) and PBMC ( E ) nuclear extract over the range 0–0.5 μg/well for the HepG2 nuclear extract ( r 2 = 0.99) and over the range 0–0.63 μg/well for the peripheral blood mononuclear cell nuclear extract ( r 2 = 0.99). Data shown represent the mean ± SD of triplicate technical replicates.

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay, Incubation, Concentration Assay

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Sensitivity and reproducibility of DNA repair enzyme activity assays

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay, Intra Assay, Inter Assay

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Within and between cell line variability in DNA repair enzyme activity of HepG2 and Caco-2 cells. ( A ) Uracil DNA glycosylase activity was higher in Caco-2 nuclear extract than HepG2 nuclear extract. ( B ) No significant differences were found in apurinic/apyrimidinic site incision activity between the two cell lines. ( C ) DNA polymerase activity was lower in Caco-2 nuclear extract than HepG2 nuclear extract. ( D ) DNA ligase activity was lower in Caco-2 nuclear extract than HepG2 nuclear extract. Bars indicate mean ± SD of six independent replicates. The symbols * and *** indicate significant differences between the enzyme activities of the cell lines at P < 0.05 and P < 0.001, respectively (based on two-tailed unpaired t -tests).

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay, Two Tailed Test

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Base excision repair enzyme activity is correlated between some, but not all, enzymes of the pathway in nuclear extracts of peripheral blood mononuclear cells from different individuals. ( A ) Uracil DNA glycosylase (UDG) activity was positively correlated with apurinic/apyrimidinic (AP) site incision activity ( r = 0.61, P < 0.05). ( B ) UDG activity was not significantly correlated with DNA polymerase activity ( r = 0.58, P = 0.053). ( C ) UDG activity was positively correlated with DNA ligase activity ( r = 0.81, P < 0.001). ( D ) AP site incision activity was not significantly correlated with DNA polymerase activity ( r = 0.40, P = 0.177). ( E ) AP site incision activity was positively correlated with DNA ligase activity ( r = 0.73, P < 0.01). ( F ) DNA polymerase activity was positively correlated with DNA ligase activity ( r = 0.67, P < 0.05).

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Complete repair of an apurinic/apyrimidinic (AP) site or a tetrahydrofuran AP site analog by HepG2 nuclear extract. The data presented in ( A ) demonstrate that color development observed in the assay plate wells is directly proportional to the amount of fluorescein modified oligonucleotide retained in the wells ( r 2 = 0.99). These data were generated by (i) coating the wells of a Nunc ® Immobilizer™ plate with 0.5 pmol/well of mixtures containing varying proportions of oligonucleotides URA03 (containing a single uracil lesion) and Contr01 (lesion free control), (ii) hybridizing and ligating a complementary oligonucleotide (Loop01Aflc) to the immobilized URA03/Contr01 mixtures to form intact hairpin loop structures covalently attached to the plate at the 3′ end with a fluorescein moiety at the 5′ end, (iii) incubating the immobilized substrates with excess Escherichia coli uracil DNA glycosylase to excise the uracil from those oligonucleotide carrying them to create AP sites, (iv) subjecting the substrate to hot alkaline denaturation to convert all AP sites into single strand breaks and remove any oligonucleotides released as a result and (v) detecting fluorescein retained in the wells using anti-fluorescein horseradish peroxidase conjugate and TMB substrate. Based on this observation, percentage repair was determined by linear interpolation from the absorbance values for 0 and 100% repair control wells. ( B ) Effects of varying concentrations of HepG2 nuclear extract on the extent of complete AP site repair in the absence (open circles) and presence (closed circles) of excess T4 DNA ligase. ( C ) Comparison of extent of complete repair by HepG2 nuclear extract of an oligonucleotide complex containing an AP site that had either been pre-treated or not with excess recombinant human AP endonuclease 1. ( D ) Comparison of the extent of complete repair of an oligonucleotide containing either a normal AP site (black bars) or the tetrahydrofuran AP site analog (white bars) by 800 or 400 ng/well of HepG2 nuclear extract in the presence of either 30 μM of all four deoxynucleotides (dATP, dCTP, dGTP and dTTP) or only 30 μM dTTP. Error bars indicate mean ± SD of triplicate technical replicates.

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Modification, Generated, Control, Comparison, Recombinant

Journal: Nucleic Acids Research

Article Title: A panel of colorimetric assays to measure enzymatic activity in the base excision DNA repair pathway

doi: 10.1093/nar/gkz171

Figure Lengend Snippet: Assay for alkyladenine DNA glycosylase (AAG) activity. Incubation with recombinant human AAG led to enzyme concentration- ( A ) and time-dependent ( B ) decreases in the amount of intact oligonucleotide substrate containing an I:T base pair following alkaline denaturation. For the time course analysis squares, circles and triangles indicate data generated with 0.1, 0.2 or 0.4 units/well, respectively. Incubation with HepG2 nuclear extract ( C ) or peripheral blood mononuclear cell pooled extract ( D ) also caused concentration-dependent decreases in the amount of intact substrate retained in the wells. The apparent AAG activity was directly proportional to the sample concentration over the range 0–10 μg/well for HepG2 ( E ) and peripheral blood mononuclear cell ( F ) nuclear extracts ( r 2 = 0.94 in both cases). Data shown represent the mean ± SD of triplicate technical replicates except for time course data for which duplicates were analyzed.

Article Snippet: Recombinant T4 DNA ligase, 3′ → 5′ exonuclease minus Klenow fragment of Escherichia coli DNA polymerase I (exo − Klenow), deoxynucleotide triphosphates (dNTPs) and herring sperm DNA were purchased from Promega (Southampton, UK).

Techniques: Activity Assay, Incubation, Recombinant, Concentration Assay, Generated