Journal: Nature communications

Article Title: UBE2D3 facilitates NHEJ by orchestrating ATM signalling through multi-level control of RNF168.

doi: 10.1038/s41467-024-49431-6

Figure Lengend Snippet: Fig. 6 | UBE2D3 promotes KAP1 phosphorylation and telomere NHEJ in a PP2A- dependent manner. a Immunoblotting for pKAP1 (S824) in TRF2ts MEFs trans- duced with control, Ube2d3 and/or two independent Ppp2ca shRNAs at 32 °C or after 3 h at 37 °C. Representative blots from 2 independent experiments. b Quantification of chromosome fusions in TRF2ts MEFs transduced with control, Ube2d3 and two independent Ppp2ca shRNAs, upon 24 h of telomere uncapping. Two independent experiments are shown. c PP2A activity assays with immuno- precipitated PP2A from TRF2ts MEFs transduced as indicated and cultured at 32 °C or for 3 h at 37 °C to induce telomere uncapping (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). Immunoblots of input and immunopre- cipitates are shown in Supplementary Fig. 8e. d PP2A phosphatase activity assay with immunoprecipitated PP2A-alpha (PP2Ac) from RNF168 mutant human cells (RIDDLE) with and without expression of ectopic HA-RNF168. Corrected for the amount of immunoprecipitated PP2A-alpha. Cells were untreated or harvested 30 min after irradiation with 3 Gy (n = 3 independent experiments; mean ± SEM; two-tailed Student’s t-test). The different symbols (dot, square and triangle)

Article Snippet: Primary antibodies used were against UBE2D3 (Y-25, sc-100618, SCBT, 1:500; 11677-1-AP, Proteintech, 1:500; 4330S, CST, 1:500 and A615, Boston Biochem, 1:2000), KAP1 (22553, Abcam, 1:1000), phospho-Kap1 S824 (A300 767A, Bethyl, 1:1000), 53BP1 (NB100-305, Novus, 1:500 and A300-272A, Bethyl, 1:2000), phospho-ATMS1981 (4526, CST, 1:1000), phospho-H2AX S139 (5636, Millipore, 1:1000), CHK2 (611570, BD, 1:500), c-myc (9E10, sc-40, SCBT, 1:250), HA (MMS101R, Covance, 1:1000), TRF2 (NB110-57130, Novus, 1:500), RNF8 (sc133971, SCBT, 1:250), MAD2L2 (14, sc-135977, SCBT, 1:500), GFP IgG fraction (A11122, Thermo Fisher Scientific, 1:1000), FLAG M2 (F1804, Sigma-Aldrich, 1:1000), Histone H3 (ab1791, Abcam, 1:10,000), hRNF168 (ABE367, Millipore, 1:500), hRNF168 (ABE467, Merck-Millipore, 1:1000), mRnf168 (gift from D. Durocher, 1:1000), hRIF1 (A300569A, Bethyl, 1:1000), mRIF1 (gift from S. Boulton and R. Chapman, 1:1000), Ligase 4 (H-300, sc-28232, SCBT, 1:300; NB110-57379, Novus, 1:500), FK2 (04-263, Millipore, 1:2000), HP1α (2616S, CST, 1:1000), Ubiquitin (P4D1, sc-8017, SCBT, 1:1000), HDAC1 (PA1-860, Thermo Fisher Scientific, 1:1000), PP2A C subunit, clone 1D6 antibody (05-421, Sigma-Aldrich/Millipore, 1:500), CDK4 (C-22, sc-260, SCBT, 1:500), HSP90 α/β (H-114, sc-7947, SCBT, 1:1000), γ-tubulin (T6557, SigmaAldrich, 1:10,000) β-actin (A5316, Sigma-Aldrich, 1:10,000), β-catenin (610154, BD, 1:10,000) and GAPDH (PA1-987, Thermo Fisher Scientific, 1:1000).HSP90α/β, γ-tubulin,β-actin,β-catenin andGAPDHwere used for loading controls.

Techniques: Phospho-proteomics, Western Blot, Control, Transduction, Activity Assay, Cell Culture, Two Tailed Test, Phosphatase Assay, Immunoprecipitation, Mutagenesis, Expressing, Irradiation

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 is the E3 ligase for ADP-ribosylated AR. A, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) in PC3-AR cells with siRNA knockdowns (total 4-day knockdown) of the selected relevant E3 ligases (DTX1, DTX2, DTX4, HUWE1, RNF146, SPOP, TRIP12 and UBR5), treated with R1881 for 21 hr before cell harvest. The AR/TUB and AR-ADPr/AR ratios for each lane are presented below the blot. B, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR cells with siDTX2 knockdown, treated with R1881 for times indicated on the panel. The AR-ADPr/AR ratio for each lane is presented below the blot. C, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL and siDTX2 cell extracts and AF1521 bound fraction. Cell extracts from PC3-AR siCTRL and siDTX2 cells treated with R1881 for 6 hr were combined with AF1521 beads for the enrichment of ADP-ribosylated proteins. D, Immunoblot detection of Flag-AR and AR-ADPr in PC3-AR siCTRL/siDTX2 and PC3-AR HA- PARP7 cell extracts and GSH beads bound fraction. Cell extracts from PC3-AR siCTRL/siDTX2 and PC3-AR HA-PARP7 cells treated with R1881 for 6 hr were combined with GSH beads loaded with GST- DTX2-RD or GST-DTX2-RD mut for the enrichment of proteins recognized by DTX2 DTC domain. E, Diagrams of DTX2-RD and DTX2-RD mut . Three loss of function mutations in the DTC domain of DTX2-RD mut (S568A, H582A, and H594A) are indicated with red asterisk. F, Schematic diagram of AR protein preparation as a substrate for biochemical reactions. Cell extracts from PC3-AR siDTX2 cells treated (left) or untreated (right) with R1881 for 6 hr were combined with M2 beads for immunoprecipitation. The purified protein from the preparation with R1881 treatment was used for experiments in panels G and H, and the purified protein from the preparation without R1881 treatment was used only in panel H. G, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (panel F, left). The ubiquitylated products (Ub product, red bracket) are labeled for Flag-AR and AR-ADPr detection. All reactions contained AR-ADPr (R1881 treated samples), ATP, Ub, E1 and E2. For DTX2-RD status (dropout or DTX2-RD mut ), refer to labels. H, Immunoblot detection of Flag-AR, AR-ADPr, and GST-DTX2-RD from the ubiquitylation assay on AR protein prepared with siDTX2 transfection, and with or without R1881 treatment (panel F). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The dropouts of the ubiquitylation assay components (Ub, E1, E2, and 30°C incubation) are indicated on the labels. The T7-Ubiquitin (T7-Ub) was detected by Ponceau staining. Lane numbers are indicated below the blot. I, Bar plots showing the results of an RT-qPCR experiment in PC3-AR siCTRL/siDTX2 cells untreated (grey), treated with R1881 (purple), and cotreated with R1881 and RBN2397 (blue). The y-axis represents the relative expression normalized to the GUS housekeeping gene, and the x-axis represents the siRNA used. The p-values from the Welch’s t-test for comparisons between corresponding conditions in siCTRL and siDTX2 are indicated on the plots. Error bars represent standard deviation (n = 3; n represents number of biological replicates).

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Knockdown, Immunoprecipitation, Purification, Ubiquitin Assay, Labeling, Transfection, Incubation, Staining, Quantitative RT-PCR, Expressing, Standard Deviation

Journal: bioRxiv

Article Title: Parp7 generates an ADP-ribosyl degron that controls negative feedback of androgen signaling

doi: 10.1101/2024.12.21.629908

Figure Lengend Snippet: DTX2 conjugates ubiquitin to AR through ADP-ribose A, Immunoblot detection of Fluorescein (FITC) and T7-Ubiquitin (T7-Ub) from the ubiquitylation assay on FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides. The labels indicate from the top: the substrate used (FITC-AR(C284) or FITC-AR(C284 ADPr ) peptides), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay (all reactions contained ATP, T7-Ub, E1 and E2, for DTX2-RD dropout, refer to labels), and the post- ubiquitylation assay treatments (NUDT16 or Mg 2+ buffer). Lane numbers are indicated below the blot. B, Schematic diagram representing FITC-AR(C284 ADPr ) peptide conjugated to ubiquitin (Ub). Indicated with red scissors are bonds within the ADP-ribose structure cleaved by NUDT16 and USP2. C, Schematic of the ubiquitylation assay workflow for panels D and E. D, Immunoblot detection of Flag-AR and AR-ADPr (by FL-AF1521) from the ubiquitylation assay on AR protein prepared with siDTX2 transfection and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post-ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. E, Immunoblot detection of Flag-AR and AR-ADPr from the ubiquitylation assay on AR protein prepared with siDTX2 and R1881 treatment (refer to panel 6f for sample preparation workflow). The ubiquitylated products (Ub product) are labeled in red for Flag-AR and AR-ADPr detection. The labels separated by black lines indicate sequential steps from top to bottom. From the top, the labels indicate the substrate used (AR-ADPr), pre-ubiquitylation assay treatments (NUDT16), the ubiquitylation assay, and the post- ubiquitylation assay treatments (USP2-CD, NUDT16, or Mg 2+ buffer). Lane numbers are indicated below the blot. F, Scatter plots depicting the correlation between PARP7 (left) or DTX2 (right) mRNA expression and the response to androgen pathway activity calculated by PARADIGM in primary prostate cancer patients from TCGA-PRAD cohort. Each dot represents one patient (n = 478, n represents number of patients). Pearson correlation coefficients and corresponding p-values are indicated on the plots. G, Kaplan-Meier plot depicting progression-free interval (PFI) in primary prostate cancer patients from the TCGA-PRAD cohort, stratified by PARP7 expression levels. The red line represents patients with high PARP7 expression (top 25%), and the green line represents patients with low PARP7 expression (bottom 25%). The X-axis represents time (days), and the Y-axis represents the progression-free interval probability. The interval distributions were compared using the log-rank test, with the p-value indicating statistical significance. Dotted lines represent the 95% confidence interval.

Article Snippet: Ubiquitylation assays were performed at 30°C for 30 min with 1 mM ATP, 100 g/ml Ub (T7-Ub or bovine Ub (Sigma U6253)), 5 g/ml each of UB E1 (R&D Systems E-304) and UB E2 (His-UbcH5C, R&D Systems E2-627), and 20 g/ml GST-DTX2 RD in the buffer E (20 mM Tris-HCl pH 7.5, 50 mM NaCl, 2 mM MgCl2, 1 mM DTT, 1 µg/ml each of A/L/P and 0.1 mg/ml BSA).

Techniques: Western Blot, Ubiquitin Assay, Transfection, Sample Prep, Labeling, Expressing, Activity Assay