Journal: Journal of medicinal chemistry

Article Title: From DNA-Encoded Library Screening to AM-9747 : An MTA-Cooperative PRMT5 Inhibitor with Potent Oral In Vivo Efficacy.

doi: 10.1021/acs.jmedchem.4c03101

Figure Lengend Snippet: Figure 7. (A) AM-9747 viability in HCT116 cellular assay (blue circles, MTAP-WT; red squares MTAP-del). Viability was measured by a CellTiter-Glo assay, and cellular MTA-selectivity was determined as (HCT116-WT IC50/MTAP-del IC50). (B) HCT116-WT and MTAP-del global SDMA levels were assessed by an in-cell imaging assay after 3 days of treatment with AM-9747.

Article Snippet: Cells were then stained with an anti-SDMA polyclonal antibody (CST#13222S at 1:2000 dilution) in a wash buffer and incubated at RT for 2 h. Cells were washed with 200 mL of Wash Buffer and stained with secondary antibodies (goat antirabbit-alexa-488 Cat #A11034, Invitrogen at 1:2000 dilution) and Hoechst 33342 DNA dye (1:5000 dilution) for 1 h at RT in the dark.

Techniques: Glo Assay, Imaging

Journal: Nucleic Acids Research

Article Title: PRMT5-mediated arginine methylation of TDP1 for the repair of topoisomerase I covalent complexes

doi: 10.1093/nar/gky291

Figure Lengend Snippet: TDP1 is methylated at R361 and R586 by PRMT5. ( A ) Schematic representation of human TDP1 showing the arginine dimethylation sites (R361 and R586), the S81-phosphorylation site, the K111-SUMOylation site and the catalytic residues (HKN motifs). Alignment of TDP1 sequences spanning R361 and R586 (highlighted in grey boxes) from human ( Homo sapiens ), monkey ( Macaca mulatta ), cattle ( Bos taurus ), rat ( Rattus norvegicus ), mouse ( Mus musculus ) demonstrates their phylogenetic conservation. ( B ) HCT116 cells ectopically expressing GFP-TDP1 were treated with or without CPT (5 μM, 3 h). GFP-TDP1 was immunoprecipitated using anti-GFP antibody and the immune complexes were blotted with SDMA-specific antibody. The same blot was stripped and reprobed with anti-GFP antibody to show equal loading. ( C ) HCT116 cells ectopically expressing GFP-TDP1 were treated with IR (10 Gy). Cells were analyzed 3 h after irradiation. GFP-TDP1 was immunoprecipitated using anti-GFP antibody and the immune complexes were blotted with SDMA-specific antibody. The same blot was stripped and reprobed with anti-GFP antibody to show equal loading. ( D ) Detection of arginine methylation of TDP1 at R361 and R586. The GFP-tagged TDP1 constructs: wild-type (GFP-TDP1 WT ), single-mutants for arginine methylation sites: GFP-TDP1 R361K and GFP-TDP1 R586K , and the double-mutant R361K + R586K [KK] were ectopically expressed in HCT116 cells, treated as indicated with CPT (5 μM, 3 h). GFP-TDP1 variants were immunoprecipitated using anti-GFP antibody and the immune complexes were blotted with anti-SDMA-specific antibody. The same blot was stripped and reprobed with anti-GFP antibody to show equal loading. Control immunoprecipitation with anti-IgG demonstrates the specificity of the reactions. Migration of protein molecular weight markers (kDa) is indicated at right. ( E ) PRMT5 depletion abrogates the symmetric dimethylation of arginine residues on TDP1. HCT116 cells were transfected with PRMT5 or control (Ctr) siRNA, then transfected 48 h later with a GFP-tagged human TDP1 construct (GFP-TDP1 WT ). Following CPT treatment (5 μM, 3 h), ectopic GFP-TDP1 was immunoprecipitated using anti-GFP antibody and the immune complexes were blotted with SDMA specific antibodies. The same blot was stripped and reprobed with anti-GFP antibody. Aliquots (10%) of the input show the level of PRMT5 knockdown, and GFP-TDP1 prior to immunoprecipitation. Electrophoretic migration of protein molecular weight markers (kDa) is indicated at right. ( F ) In vitro methylation assay with flag-tagged PRMT5 immunoprecipitated from HCT116 cells using anti-flag antibody with unlabeled S-adenosylmethionine (SAM). The substrates were recombinant His-tagged TDP1: wild-type (WT) and double-mutant for the R361 and R586 methylation sites (KK). The same blot was stripped and reprobed with anti-TDP1 antibody showing the amount of substrate in each reaction. Migration of protein molecular weight markers (kDa) is indicated at right.

Article Snippet: Rabbit polyclonal anti-symmetric dimethyl arginine (SDMA) (SYM10 and SYM11), anti-PRMT5 (07-405), anti-PRMT9 (MABE1112) and mouse monoclonal anti-γH2AX (05-636) antibodies were purchased from Millipore, USA.

Techniques: Methylation, Phospho-proteomics, Expressing, Immunoprecipitation, Irradiation, Construct, Mutagenesis, Control, Migration, Molecular Weight, Transfection, Knockdown, In Vitro, Recombinant

Journal: Nucleic Acids Research

Article Title: PRMT5-mediated arginine methylation of TDP1 for the repair of topoisomerase I covalent complexes

doi: 10.1093/nar/gky291

Figure Lengend Snippet: Replication-coupled DNA damage induces TDP1 arginine methylation and PRMT5-dependent Top1cc repair. ( A ) TDP1 methylation induction by replication DNA damage. HCT116 cells were transfected with a GFP-tagged human TDP1 construct (GFP-TDP1 WT ). Cells were pre-treated with 1 μM aphidicolin (APH) for 15 min or 10 μM DRB for 1 h. Following CPT treatment (5 μM, 3 h), ectopic GFP-TDP1 was immunoprecipitated using anti-GFP antibody and the immune complexes were blotted with SDMA specific antibodies. The same blot was stripped and reprobed with anti-GFP antibody. ( B ) Densitometry analysis of arginine methylation of TDP1 (SDMA-TDP1) shown in panel A normalized against GFP TDP1. ( C ) PRMT5 depletion enhances replication-associated γH2AX. Confocal immunofluorescence microscopic analysis of CPT (5 μM, 3 h) induced γH2AX in control and PRMT5-depleted HCT116 cells pretreated with APH (1 μM, 15 min), DRB (10 μM, 1 h), or, both (APH +DRB, 1h) as indicated. PRMT5 and γH2AX are shown in red and green respectively. Nuclei were stained with DAPI (blue). ( D ) Quantification of replication and transcription associated CPT-induced γH2AX intensity per nucleus obtained from confocal immunofluorescence microscopy were calculated for 20–30 cells (calculated value ± S.E.M.) (h). APH induced reduction (fold change) in CPT-induced γH2AX intensity in PRMT5 proficient and PRMT5 depleted cells are indicated. Asterisks denote significant difference (** P < 0.001; t test) in CPT-induced γH2AX intensity between control and PRMT5 depleted cells.

Article Snippet: Rabbit polyclonal anti-symmetric dimethyl arginine (SDMA) (SYM10 and SYM11), anti-PRMT5 (07-405), anti-PRMT9 (MABE1112) and mouse monoclonal anti-γH2AX (05-636) antibodies were purchased from Millipore, USA.

Techniques: Methylation, Transfection, Construct, Immunoprecipitation, Immunofluorescence, Control, Staining, Microscopy