Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) is highly expressed in oesophageal squamous cell carcinoma (OSCC) tissues and linked to tumour stage and poor prognosis. (A) Volcano plot showing gene expression differences between cisplatin‐resistant and parental groups in GSE169337. (B) Kaplan–Meier survival analysis of OSCC patients with different PADI4 levels using The Cancer Genome Atlas (TCGA) data. (C) Immunohistochemical (IHC) analysis comparing PADI4 expression in OSCC tissues to normal oesophageal tissues. (D) IHC analysis of PADI4 expression across TNM stages in OSCC. (E, F) Kaplan–Meier analysis of overall and recurrence‐free survival in OSCC patients with varying PADI4 expression from IHC data. (G) PADI4 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (H) PADI4 knockdown markedly inhibited the migration ability of ECA109 and KYSE150 cells indicated by transwell assay.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Gene Expression, Immunohistochemical staining, Expressing, Knockdown, Colony Assay, Migration, Transwell Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Clinical characteristics of patients and expression of peptidylarginine deiminase 4 (PADI4).

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Expressing

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Regression analysis of oesophageal squamous cell carcinoma (OSCC) patients.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques:

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Cancer stem cell (CSC)‐like phenotypes and cisplatin resistance of oesophageal squamous cell carcinoma (OSCC) cells were suppressed by peptidylarginine deiminase 4 (PADI4) knockdown. (A) Comprehensive analysis of PADIs and the stemness across 33 tumour types within the The Cancer Genome Atlas (TCGA) database. (B) Correlation between PADI4 and stemness markers (CD133, LGR5, Nanog, OCT4 and CD24) in GSE23400. (C, D) qPCR showed PADI4 knockdown significantly reduced CD133 and Nanog mRNA levels in ECA109 (C) and KYSE150 (D) cells. (E, F) Western blot revealed PADI4 knockdown significantly decreased CD133 and Nanog protein levels in ECA109 (E) and KYSE150 (F) cells. (G, H) Western blot showed PADI4 plasmids significantly increased CD133 and Nanog protein levels in ECA109 (G) and KYSE150 (H) cells. (I) PADI4 knockdown significantly reduced sphere formation in ECA109 and KYSE150 cells. (J, K) Downregulation of PADI4 enhanced the resistance of ECA109 (J) and KYSE150 (K) cells to cisplatin through CCK8 assay. (L, M) Limiting dilution assays showing the self‐renewing capacity of PADI4 downregulated in CD133‐positive cells of ECA109 and KYSE150.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Knockdown, Western Blot, CCK-8 Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) interacts with protein arginine methyltransferase 2 (PRMT2) and maintains stability of PRMT2. (A) Mass spectrometry analysis identified PRMT2 bound with PADI4. (B) Immunostaining of ECA109 cells transfected with PADI4‐HA and PRMT2‐Flag was performed using anti‐HA and anti‐Flag antibodies, with confocal microscopy revealing the subcellular localization of PADI4‐Flag (green), PRMT2 (red) and DAPI (blue, nucleus marker). (C, D) Endogenous cimmunoprecipitation was used to study the interaction between PADI4 and PRMT2 in ECA109 and KYSE‐150 cells after transformed into plasmid of PADI4‐HA. (E, F) PRMT2 protein level in PADI4‐knockdown cell lines. (G, H) PRMT2 protein level in PADI4‐upregulated cell lines. (I) PRMT2 protein degradation levels were measured in treat with cycloheximide (CHX) (10 µg/mL) over specified time intervals in PADI4‐KO ECA109 cells. (J) Lysates from cells co‐transfected with His‐Ub, Flag‐PRMT2 and PADI4‐HA underwent immunoprecipitation with a Flag antibody, followed by immunoblotting with an anti‐His antibody in HEK 293T cells. DAPI, 4′,6‐Diamidino‐2‐phenylindole.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Mass Spectrometry, Immunostaining, Transfection, Confocal Microscopy, Marker, Transformation Assay, Plasmid Preparation, Knockdown, Immunoprecipitation, Western Blot

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) citrullinates protein arginine methyltransferase 2 (PRMT2) to maintain the stability of PRMT2. (A) PADI4‐HA overexpression was immunoprecipitated using Flag‐PRMT2 antibodies, and immunoblotted with modified‐citrulline antibodies. (B) Recombinant PADI4 was immunoprecipitated with a Flag‐PRMT2 antibody in calcium presence, and reaction products were analysed by western blot with modified‐citrulline antibody. (C) The amino acid sequence of PRMT2 in different species is highly conserved at R312 and R397. (D) Two PRMT2 mutant plasmids and transfected into HEK 293T cells to perform immunoprecipitation experiments to investigate citrullination. (E) ECA109 cells were analysed by western blot after 8‐h treatment with varying doses of BB‐Cl‐Amidine. (F) In ECA109 cells treated with GSK484, PRMT2's half‐life was shown by using cycloheximide (CHX) to block protein synthesis. (G) Immunoprecipitating PRMT2 with an anti‐PRMT2‐Flag antibody and probing for polyubiquitination with an anti‐ubiquitin antibody revealed a significant increase in polyubiquitinated PRMT2 protein after GSK484 treatment. (H, I) KYSE150 (H) and ECA109 (I) cells were analysed of cancer stem cell (CSC) markers by western blot after 24‐h treatment with varying doses of GSK484.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Over Expression, Immunoprecipitation, Modification, Recombinant, Western Blot, Sequencing, Mutagenesis, Transfection, Blocking Assay, Ubiquitin Proteomics

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) acts as a positive regulator of inhibitors of DNA binding (IDs). (A, B) The mRNA and protein levels of cancer stem cell (CSC) markers in PRMT2 knockdown ECA109 cells were also assessed by qPCR and western blot. (C) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of CSC markers were measured using western blot. (D, E) The mRNA and protein levels of ID1, ID2 and ID3 in PRMT2 knockdown ECA109 cells were assessed using qPCR and western blot. (F) In ECA109 cells transfected with PRMT2 plasmids, the protein levels of ID1, ID2 and ID3 were measured using western blot. (G, H) The mRNA and protein levels of ID1, ID2 and ID3 in peptidylarginine deiminase 4 (PADI4) knockdown and ECA109 cells were assessed using real‐time PCR and western blot. (I) The protein levels of ID1, ID2 and ID3 in ECA109 cells transfected with PADI4 plasmids were measured. (J) H3R8me2a expression was analysed via western blot in PADI4‐transfected ECA109 cells. (K, L) ChIP assay revealed H3R8me2a enrichment at the human ID1 and ID2 promoter in ECA109 cells. (M, N) Cell viability was assessed using a CCK8 assay after 48‐h treatment with cisplatin in ECA109 (M) and KYSE150 cells (N). (O) PRMT2‐KO cells showed reduced sphere formation in ECA109 and KYSE150 cells. (P, Q) GSK484 reduction in the expression levels of ID1, ID2 and ID3 with different concentration in ECA109 and KYSE150 cells. (R, S) Limiting dilution assays showing the self‐renewing capacity of PRMT2 downregulated in ECA109 (R) and KYSE150 (S) CD133‐positive cells. (T) PRMT2 knockdown markedly inhibited the proliferation ability of ECA109 and KYSE150 cells indicated by colony formation assay. (U) Immunihistochemical (IHC) analysis comparing PRMT2 expression in oesophageal squamous cell carcinoma (OSCC) patients tumour tissues to adjacent normal oesophageal tissues. (V) Correlation between PADI4 and PRMT2 in OSCC patients tumour tissues.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Binding Assay, Knockdown, Western Blot, Transfection, Real-time Polymerase Chain Reaction, Expressing, CCK-8 Assay, Concentration Assay, Colony Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Protein arginine methyltransferase 2 (PRMT2) citrullinate disrupts the interaction between USP7 and PRMT2. (A) HEK 293T cells were co‐transfected with PRMT2‐Flag and PADI4‐HA plasmids, and USP7 levels were assessed via western blot. (B) Co‐transfect HEK 293T cells with PADI4‐Flag and USP7‐HA, treat with GSK484 (20 µM) for 24 h, and use western blot to evaluate PRMT2 levels. (C) HEK 293T cells were co‐transfected with PRMT2‐WT‐Flag, PRMT2‐R312E‐Flag and PRMT2‐R397E‐Flag plasmids, and USP7 levels were assessed via western blot. (D) HEK 293T cells were co‐transfected with HA‐USP7, His‐Ub, PRMT2‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, then treated with MG132 (20 µM) for 5 h before harvesting to assess PRMT2‐Flag ubiquitination levels. (E) HEK 293T cells were co‐transfected with HA‐USP7, PRMT2‐WT‐Flag, PRMT2‐R397E‐Flag and PRMT2‐R312E‐Flag, and then use western blot to evaluate Flag levels. (F) Western blot was used to analyse ID1, ID2 and ID3 expression in HEK 293T cells transfected with PRMT2‐WT, PRMT2‐R397E and PRMT2‐R312E. (G) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate resistance of ECA109 cells to cisplatin. (H) Peptidylarginine deiminase 4 (PADI4) knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (I) ECA109 cell line transfected plasmid overexpressing PRMT2 or PRMT2‐R312E to evaluate the sphere formation. (J) PADI4 knockdown reduced sphere formation in ECA109 cells and PRMT2 restores this effect. (K) Downregulation of PADI4 enhanced the resistance of ECA109 cells to cisplatin through CCK8 assay and PRMT2 restores this effect.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: Transfection, Western Blot, Ubiquitin Proteomics, Expressing, Plasmid Preparation, Knockdown, CCK-8 Assay

Journal: Clinical and Translational Medicine

Article Title: PADI4 facilitates stem‐like properties and cisplatin resistance through upregulating PRMT2/IDs family in oesophageal squamous cell carcinoma

doi: 10.1002/ctm2.70272

Figure Lengend Snippet: Peptidylarginine deiminase 4 (PADI4) facilitates tumour growth and metastasis in vivo, while GSK484 exhibits a synergistic effect with cisplatin in vivo. (A) A schematic diagram illustrating the treatment protocol for mice in this study is presented. (B, C) Images of excised subcutaneous tumours from mice treated with shPADI4, GSK484 and cisplatin, and the combination of GSK484 and cisplatin are shown. (D) Immunohistochemical (IHC) analysis of PADI4 protein expression in excised tumours from the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is presented. (E) The volume of subcutaneous tumours in the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is depicted. (F) The weight of subcutaneous tumours in the shPADI4, GSK484, cisplatin and GSK484 plus cisplatin treatment groups is reported. (G) HE staining revealed oesophageal squamous cell carcinoma (OSCC) metastases in the lung tissues. (H) The schematic diagram of the underlying mechanism in this study was shown.

Article Snippet: A total of 1000 ng of recombinant PADI4 (HY‐P70990, MedChemExpress Technology) was incubated with either Flag‐PRMT2 wild‐type or mutant in a 200 μL buffer containing 5 mM DTT, 10 mM CaCl 2 , and 50 mM NaCl at 37°C for 4 h. The presence of protein‐bound citrulline was subsequently detected using a modified citrulline antibody, specifically the Anti‐Citrulline antibody (StressMarq Biosciences, SMC‐501D).

Techniques: In Vivo, Immunohistochemical staining, Expressing, Staining

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) Top: Protein domain architecture of human FAN1 indicating the location of the MIP box. PIP, proliferating cell nuclear antigen–interacting peptide box; UBZ, ubiquitin-binding zinc finger; SAP, SAF-A/B, Acinus, and PIAS; TPR, tetratricopeptide repeat; NUC, nuclease. Bottom: Sequence alignment of the FAN1 orthologs with the MIP box of human EXO1 and BLM. ( B ) Dissociation constants ( K d ) indicating the binding affinity of MLH1 CTD for the indicated peptides from FAN1, EXO1, and BLM as determined by ITC. NI, no interaction (weak and constant signal under the condition used). ( C ) Immunoblots of FLAG-M2 affinity resin immunoprecipitations (IPs) of extracts from HEK293 cells transfected either with empty vector (e.v.), FLAG-FAN1 wild type (wt), or the variant constructs. The Y128A/F129A mutation is denoted as MIP*. ( D ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells transfected with the indicated FLAG-FAN1 constructs. ( E ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells cotransfected with FLAG-FAN1 and vectors expressing hemagglutinin (HA)–tagged dominant-negative (dn) forms of CDK1 or CDK2. ( F ) Immunoblots of green fluorescent protein (GFP)–Trap IPs of extracts from HeLa GFP-FAN1 cells synchronized in mitosis by thymidine-nocodazole block. Mitotic cells were replated in fresh medium for the indicated time periods. The relative ratios of pS126 to FAN1 and MLH1 to FAN1 in GFP-Trap samples were quantified by densitometry using ImageJ. (C to F) The antibodies used are shown on the left.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Ubiquitin Proteomics, Binding Assay, Sequencing, Western Blot, Transfection, Plasmid Preparation, Variant Assay, Construct, Mutagenesis, Expressing, Dominant Negative Mutation, Blocking Assay

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) Top: Protein domain architecture of human FAN1 indicating the location of the MIP box and MIM. Bottom: Sequence alignment of FAN1 orthologs with the putative MIM of human MLH3, PMS2, and PMS1. ( B ) ITC was used to determine the K d indicating the binding affinity of MLH1-CTD for the indicated peptides. ( C ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells transfected either with e.v. or indicated FLAG-FAN1 expression constructs. The L155A/L159A mutation is denoted as MIM*. ( D ) Immunoblots of FLAG-M2 affinity resin IPs of extracts from HEK293 cells transfected either with e.v. or indicated FLAG-FAN1 expression constructs. ( E and F ) Recombinant MutLα (E) or MutLγ (F) (200 nM) was subjected to pull-down reactions using the indicated recombinant GST-FAN1 (amino acids 118 to 177) variants. (C to F) The antibodies used are shown on the left.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Sequencing, Binding Assay, Western Blot, Transfection, Expressing, Construct, Mutagenesis, Recombinant

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) PLA was used to evaluate FAN1-MLH1 association in U2OS GFP-FAN1 wt and U2OS GFP-FAN1 MIP*/MIM* cells mock-treated or treated with MMC (150 ng/ml) for 24 hours. Representative images are shown. Scale bars, 10 μm. Scatterplot displays quantification of the PLA signals per nucleus from at least 100 cells. Data display the means ± SD from three independent experiments. Statistical significance was calculated by unpaired t test. ** P < 0.01; ns, not significant. ( B ) Schematic representation of human FAN1, highlighting positions of mutations used, including MIP*, MIM*, dimerization-defective (dim*), and nuclease-defective (nd*) FAN1 variants. ( C and D ) Immunoblots of GFP-Trap IPs of extracts from indicated U2OS GFP-FAN1 cells. The antibodies used are shown on the left. ( E ) Clonogenic survival assay of the indicated U2OS GFP-FAN1 cells exposed to increasing doses of MMC. Viability of untreated cells was defined as 100%. Data are presented as the means ± SEM.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Western Blot, Clonogenic Cell Survival Assay

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) QIBC analysis of GFP-FAN1 foci in U2OS GFP-FAN1 cells mock-treated, treated with MMC (20 ng/ml) for 24 hours, or MMC-treated and then released for 72 hours. Color-coded scatterplots indicate the number of GFP-FAN1 foci per nucleus. ut, untreated. ( B ) Same cells as in (A) were pulse-labeled with ethynyl deoxyuridine (EdU) during the last 30 min before harvesting and subjected to the Click-IT reaction. Cell cycle distribution was evaluated by QIBC using the 4′,6-diamidino-2-phenylindole (DAPI) and EdU signals (fig. S5C). ( C ) QIBC of RPA2 foci in U2OS GFP-FAN1 cells mock-treated, treated with MMC (20 ng/ml) for 24 hours, or MMC-treated and then released for 72 hours. Color-coded scatterplots indicate the number of RPA2 foci per nucleus. A.U., arbitrary units. ( D ) Same cells as in (C) were treated with MMC (300 ng/ml) for 24 hours, and lysates were analyzed by immunoblotting using the indicated antibodies. Asterisk indicates hyperphosphorylated form of RPA2.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Labeling, Western Blot

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: ( A ) Scheme of the slipped (CTG)30/(CAG)50 substrate. ( B ) The (CAG)20 slip-out substrate was incubated with extracts from U2OS GFP-FAN1 cells expressing indicated FAN1 variants. Repair efficiency was quantified by densitometric analysis of Southern blots. Values represent the mean of three independent experiments. Error bars represent ±SD. Statistical significance was calculated by ordinary one-way analysis of variance (ANOVA) test followed by Tukey’s multiple comparisons. ** P < 0.005, *** P < 0.0005, **** P < 0.00005. ( C ) HeLa nuclear extract was incubated with FAN1-derived 60-mer peptides (amino acids 118 to 177) containing wt or mutant MIP-MIM, immunoprecipitated using anti-FAN1 antibody, and immunoblotted with the indicated antibodies. ( D ) Extracts from HeLa cells were supplemented with increasing concentrations of FAN1 peptides and incubated with the (CAG)20 slip-out substrate. Repair efficiency was calculated as described in (B). ( E ) Extracts of U2OS GFP-FAN1 wt and U2OS GFP-FAN1 MIP*/MIM* cells were supplemented with indicated FAN1 peptides and incubated with the (CAG)20 slip-out substrate. Repair efficiency was calculated as described in (B).

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Incubation, Expressing, Derivative Assay, Mutagenesis, Immunoprecipitation

Journal: Science Advances

Article Title: FAN1-MLH1 interaction affects repair of DNA interstrand cross-links and slipped-CAG/CTG repeats

doi: 10.1126/sciadv.abf7906

Figure Lengend Snippet: Summary of the FAN1-MLH1 complex interaction and a plausible model for its involvement in ICL and slip-out repair. ( A ) Scheme of the FAN1-MLH1 interaction in different states of wt or mutated FAN1. Protein orientation is unknown and arbitrarily presented for ease. ( B ) Left: FAN1 is subjected to CDK-mediated phosphorylation at S126 located within the MIP box. Right: Regulation of FAN1-MLH1 interaction through the cell cycle and in response to ICL damage. ( C ) Top: The FAN1-MLH1 complex localizes to ICL damaged chromatin to preserve genome stability and ensure cell viability. In cells, devoid of the FAN1-MLH1 interaction aberrant ICL repair and cell death ensues. Inactivation of FAN1’s endo- and exonuclease activities by mutating D960A or inhibition of FAN1 dimerization, affecting FAN1’s endo- but not exonucleolytic activity, would alleviate the toxicity of the FAN1 MIP/MIM-mutated variant and promote cell proliferation and survival. Bottom: Inhibition of the FAN1-MLH1 interaction (MIP*/MIM*) blocks repair of slipped-DNAs. Repair defects were rescued when the MLH1-interacting–defective FAN1 was also defective either in both its endonuclease activities or in dimer formation (endo- but not exonuclease defective). Therefore, the regulatory aspects of the FAN1 binding to MLH1 aligns the pathway of ICL repair with that of slipped-DNA processing. Mutations: MIP*, Y128A/F129A; MIM*, L155A/L159A; nd*, D960A; dim*, K525E/R526E/K528E.

Article Snippet: To analyze the effect of synthetic 60-mer FAN1 peptide on repair efficiency, cell extracts were incubated in the absence or presence of either wt or mutant synthetic 60-mer FAN1 peptide (GenScript) for 30 min at RT before starting the repair reactions.

Techniques: Phospho-proteomics, Inhibition, Activity Assay, Variant Assay, Binding Assay