Journal: Nucleic Acids Research

Article Title: USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing

doi: 10.1093/nar/gkab892

Figure Lengend Snippet: Systematic screening of PARP1-dependent DUBs associated with the DDR. ( A ) Schematic representation of the high-throughput screening (HTS) strategy to identify PARP1-dependent DUBs. ( B ) Phylogenetic analysis of the six identified DUBs. The sequence-based phylogenetic tree was analyzed by Molecular Evolutionary Genetics Analysis software (MEGA6). ( C ) PAR overlay assay. Recombinant DUBs were separated by SDS-PAGE and then used in the PAR overlay assay. Iduna was used as a positive control. ( D ) Schematic illustration of functional domains of ZF-UBP DUBs. ( E ) GFP-USP39 was transfected into U2OS cells and then treated with either an ATM inhibitor (KU55933) or a PARP inhibitors (PJ34 and Olaparib). Stripe formation by USP39 was analyzed in living cells. Data represent the mean ± standard error of the mean (s.e.m.) from ten cells or more. For parametric multiple comparison, one-way analysis of variance followed by the Tukey-Kramer test was used. (*** P ≤ 0.001). Each experiment was performed independently three times. (F–H) Endogenous USP39 is accumulated at the DNA lesions. DNA lesions were induced by mIR in U2OS cells and the translocation of endogenous USP39 to the lesions was analyzed using immunostaining as indicated ( F ). Each experiment was performed independently three times. ChIP-qPCR analysis for the distribution of endogenous USP39 ( G ) and γH2AX ( H ) in FokI-induced DSBs (G and H). Data represent mean ± s.e.m. from three independent ChIP-qPCR analyses. Each dot represents the mean of one experiment. Statistical significance was determined using the Student's t -test (*** P ≤ 0.01). IgG was used as negative control. Scale bars, 5 μm.

Article Snippet: Human osteosarcoma (U2OS) and normal MRC-5 cell lines were purchased from ATCC.

Techniques: High Throughput Screening Assay, Sequencing, Software, Overlay Assay, Recombinant, SDS Page, Positive Control, Functional Assay, Transfection, Comparison, Translocation Assay, Immunostaining, ChIP-qPCR, Negative Control

Journal: Nucleic Acids Research

Article Title: USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing

doi: 10.1093/nar/gkab892

Figure Lengend Snippet: USP39 is a key factor for maintenance of genomic integrity and cell survival. ( A ) The knockdown efficacy of each USP39 si RNA or its pool ( si USP39-A, -B and -C mixture) was tested in U2OS cells. ( B ) Ablation of endogenous USP39 leads to genomic instability. USP39-depletion induced genomic instability, which was monitored by a neutral comet assay (left panel), and the level of genomic fragmentation was quantified as indicated (right panel). si RNA-targeting LIG4 was used as a positive control for the comet assay. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test ( C ) Clonogenic cell survival assay. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test. ( D and E ) Analysis of BRCA1 and 53BP1 IRIF formation. U2OS cells were transfected with control or USP39 siRNAs. Cells were fixed and stained with BRCA1 ( D ) or 53BP1 ( E ) antibodies, and nuclei were counterstained with DAPI as indicated by experimental conditions (left panel). Foci formation per cell ≥ 100 was calculated as indicated (right panel). Statistical significance was determined by the Student's t -test ( F ) Comparative analysis of HR and NHEJ repair activities in USP39 knockdown cells. si CtIP and si LIG4 were used as a positive control for HR and NHEJ repair assay, respectively. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test ( G ) GFP- si Re USP39 was generated, and its resistance against USP39 si RNA was tested. ( H ) GFP- si Re USP39 was reintroduced into cells with ablated endogenous USP39 and USP39-dependent regulation of genomic stability was monitored and quantified as described above. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test ( I ) USP39 is involved in the early response to DNA damage. USP39 si RNA and V5- si Re USP39 were transfected into U2OS cells and 48 h later, cells were treated with γ-irradiation (2 Gy). The number of γH2AX foci at DSB sites was monitored (left panel) and quantified (right panel). Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test. Data represent the mean ± s.e.m. from three hundred cells or more. All results represent at least three independent experiments. *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. n.s., not significant.

Article Snippet: Human osteosarcoma (U2OS) and normal MRC-5 cell lines were purchased from ATCC.

Techniques: Knockdown, Neutral Comet Assay, Positive Control, Single Cell Gel Electrophoresis, Clonogenic Cell Survival Assay, Transfection, Control, Staining, Generated, Irradiation

Journal: Nucleic Acids Research

Article Title: USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing

doi: 10.1093/nar/gkab892

Figure Lengend Snippet: The RG motif of USP39 is not only involved in interaction with PAR-chains but also essential for its recruitment to DNA lesions. ( A ) Schematic illustration of USP39 mutants. ( B ) Analysis of PAR-binding activity of USP39 WT or each deletion mutant. Indicated proteins were purified from insect cells and then used in PAR overlay assays. PAR-binding activity was monitored by immunoblot with the indicated antibody. (C and D) The N-terminal region of USP39 is critical for its recruitment to mIR-induced DSBs. Indicated mutants of GFP-tagged USP39 were transfected to U2OS cells, and stripe formation by USP39 WT or deletion mutants was analyzed from fixed ( C ) or living cells ( D ) as indicated. Data represent the mean ± s.e.m. from five cells. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test ( E ) Identification of tripartite RG motifs as the putative PAR-binding sites in USP39. ( F ) Recombinant USP39 WT and RG/AA mutant were subjected to the PAR overlay assay as indicated. GST or H3 was used as negative or positive controls, respectively. ( G ) Tripartite RG motifs are critical for translocation of USP39 to mIR-induced DSBs. GFP-USP39 WT or RG/AA mutant were transfected into U2OS cells and mIR-induced stripe formation was monitored in living cells (upper panel). The efficacy of translocation was quantified as indicated (lower panel). Data represent the mean ± s.e.m. from five cells or more. Statistical significance was determined by the Student's t -test. (H and I) Comparative analysis of HR ( H ) and NHEJ ( I ) repair activities in USP39 knockdowns or cells rescued by reintroduction of si Re USP39 or RG/AA as indicated. Statistical significance was determined using one-way ANOVA followed by the Tukey–Kramer test ( J ) Clonogenic cell survival assay, which was performed using the indicated experimental conditions. Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test. Scale bars, 5 μm. Data represent the mean ± s.e.m. of three independent experiments. *** P ≤ 0.001, ** P ≤ 0.01. n.s., not significant.

Article Snippet: Human osteosarcoma (U2OS) and normal MRC-5 cell lines were purchased from ATCC.

Techniques: Binding Assay, Activity Assay, Mutagenesis, Purification, Western Blot, Transfection, Recombinant, Overlay Assay, Translocation Assay, Clonogenic Cell Survival Assay

Journal: Nucleic Acids Research

Article Title: USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing

doi: 10.1093/nar/gkab892

Figure Lengend Snippet: USP39 drives PAR-seeded liquid demixing via its RG repeats in the N46 region. (A–C) USP39 WT leads to transient formation of distinct light-diffracting dark stripes via its RG motif. ( A ) USP39 WT and RG/AA mutant were transfected into U2OS cells and then monitored for translocation to DSBs using a mIR system. ( B ) The formation of USP39-mediated dark stripes was monitored in presence of a PARP inhibitor. ( C ) ATM-dependent RNF8 was used as negative control. mIR-induced dark stripe formation was monitored in bright-field. Each right panel show a 12× magnification of the last DIC images. Red lines indicate a micro-irradiated area and blue arrows point to light-diffracting dark stripes. Scale bars, 5 μm. ( D ) The N46 of USP39 is sufficient for phase separation. The N-terminal regions of GFP-USP39 (1–46, 47–98, RG/AA) were transfected into U2OS cells that were then monitored for the formation of dark stripes after mIR-induced DNA damage. Scale bars, 5 μm. Each right panel shows a 12× magnification of the last DIC image. Red lines indicate a micro-irradiated area and blue arrows point to light-diffracting dark stripes. ( E ) Endogenous USP39 regulates PAR-seeded phase separation. si USP39 or si PARG were transfected into U2OS cells and mIR-induced dark stripe formation was then monitored using bright-field microscopy. Representative images are shown in the upper panel and quantification results are shown in the lower panel. Scale bars, 5 μm. Statistical significance was determined by the Student's t -test. ( F ) USP39 WT and RG/AA mutant were incubated at RT for 24 h with or without PAR or PARG-treated PAR. Aggregate sizes were analyzed by TEM (left panel) and quantified (right panel). Statistical significance was determined using one-way ANOVA followed by the Tukey Kramer test. Scale bars, 2 μm. Data represent the mean ± s.e.m. of three independent experiments and quantification results represent the mean ± s.e.m. from five cells. *** P ≤ 0.001, n.s., not significant.

Article Snippet: Human osteosarcoma (U2OS) and normal MRC-5 cell lines were purchased from ATCC.

Techniques: Mutagenesis, Transfection, Translocation Assay, Negative Control, Irradiation, Microscopy, Incubation

Journal: Nucleic Acids Research

Article Title: USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing

doi: 10.1093/nar/gkab892

Figure Lengend Snippet: USP39 controls the recruitment of XRCC4/LIG4 to DSBs for NHEJ repair. (A and B) USP39 is key regulator of the NHEJ repair pathway by controlling the recruitment of APXL proteins (APTX, PAXX, XRCC4, and LIG4). The indicated NHEJ regulatory factors were transfected into U2OS cells along with controls or USP39-targeted si RNA. After 48 h, cells were analyzed in the mIR system and translocation efficacy was monitored ( A ) and quantified ( B ). Statistical significance was determined by the Student's t -test. Scale bars, 5 μm. Data represent the mean ± s.e.m. from five cells or more. *** P ≤ 0.001, ** P ≤ 0.01, * P ≤ 0.05. n.s., not significant.

Article Snippet: Human osteosarcoma (U2OS) and normal MRC-5 cell lines were purchased from ATCC.

Techniques: Transfection, Translocation Assay

Journal: Nucleic Acids Research

Article Title: USP39 promotes non-homologous end-joining repair by poly(ADP-ribose)-induced liquid demixing

doi: 10.1093/nar/gkab892

Figure Lengend Snippet: USP39 is a key factor in creating NHEJ complexes by interplay with PAXX, APTX, XRCC4 and LIG4. ( A ) GST-free USP39 directly interacts with recombinant GST-APTX, GST-XRCC4 and GST-LIG4, but not GST-PAXX. Indicated recombinant proteins were purified from insect cells and used in a USP39 overlay assay. GST was used as negative control. (B and C) Cell extracts from HEK293FT-expressing FLAG-tagged NHEJ factors ( B ) or FLAG-USP39 ( C ) were immunoprecipitated with anti-FLAG antibody and then analyzed by immunoblotting. ( D ) Endogenous USP39 interacts more strongly with endogenous LIG4 in the DNA-damaged state. The cell lysates derived from normal or DNA-damaged cells were immunoprecipitated with anti-USP39 antibody, and then analyzed for interaction between endogenous USP39 and endogenous LIG4. ( E–G ) Depletion of USP39 leads to failure of proper accumulation of endogenous LIG4 at DSBs. si RNA-targeting USP39 was transfected into U2OS cells and then stripe formation of endogenous LIG4 was analyzed. Yellow arrows indicate the area of signal intensity measurement ( E ). The accumulated level of γH2AX or LIG4 at the DNA lesions was analyzed by Nikon NIS software ( F ). Relative intensity of endogenous LIG4 accumulated at DSBs was monitored in cells expressing either siCtrl or siUSP39. n indicates the total cell number used in quantification ( G ). Statistical significance was determined by the Student's t -test. Scale bars, 5 μm. Data represent the mean ± s.e.m. of three independent experiments. *** P ≤ 0.001.

Article Snippet: Human osteosarcoma (U2OS) and normal MRC-5 cell lines were purchased from ATCC.

Techniques: Recombinant, Purification, Overlay Assay, Negative Control, Expressing, Immunoprecipitation, Western Blot, Derivative Assay, Transfection, Software

Journal: The Journal of Cell Biology

Article Title: Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair

doi: 10.1083/jcb.201803074

Figure Lengend Snippet: Depletion of GANP affects DNA repair without affecting the activation of the DDR. (A) Clonogenic survival assay in HeLa cells depleted of GANP and control cells treated with increasing concentration of phleomycin. The graph represents the average of three independent experiments with three technical replicates each, where the number of colonies for each concentration was normalized to the respective untreated condition. Statistical significance was calculated using the Mann–Whitney test (ns, P > 0.05; *, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (B) Western blot analysis of GANP and γH2Ax of RIPA extracts obtained from GANP-depleted cells and control cells. Numbers indicate γH2Ax levels quantified using ImageJ using γTubulin as loading control. (C) Graph represents the average of γH2Ax levels quantified using ImageJ in three independent experiments ( n = 3). (D) DNA damage measured with the neutral comet assay of GANP-depleted cells and control cells. The number of breaks are represented in tail moment as the tail length normalized to the percentage of DNA in the tail. Graph represents the average of two independent experiments with n ≥ 100 cells. Error bars represent SEM. (E and F) High-throughput screening performed to investigate the effect of GANP KD in DNA repair in HeLa cells. Graphs represent the percentage of positive cells for γH2AX intensity (E) and 53BP1 foci (F) compared with the nontreated (NT) cells. The x axis shows the corresponding siRNA used. Two different siRNAs for GANP were used (siGANP1 and siGANP2); unless otherwise specified in all the other experiments siGANP2 was used and is labeled siGANP. Black and gray boxes represent untreated cells (NO DRUG), and the time (in hours) of recovery after NCS treatment at 50 ng/ml for 15 min. A positive cell is defined as a cell having γH2AX intensity or number of 53BP1 foci higher than an arbitrary threshold defined on the 5% of nontreated cells. Graphs represent the average of two independent experiments, each of them performed in three technical replicates, with SD. Per each condition, n > 1,000 cells. Statistical significance was calculated using the ANOVA (*, P ≤ 0.05; **, P ≤ 0.01; ***, P ≤ 0.001). (G) Western blot analysis of total cell lysate obtained by RIPA extraction from cells treated with GANP siRNA and control cells. Hours represent the time of repair after NCS treatment. For details about statistical analysis, refer to the Statistical analysis section of Materials and methods. siCTRL, control siRNA; siNTarg, nontargeting siRNA (see ).

Article Snippet: Rabbit anti 53BP1 , Novus Biologicals (NB100-304) , IF (1:1,000).

Techniques: Activation Assay, Clonogenic Cell Survival Assay, Control, Concentration Assay, MANN-WHITNEY, Western Blot, Neutral Comet Assay, High Throughput Screening Assay, Labeling, Extraction

Journal: The Journal of Cell Biology

Article Title: Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair

doi: 10.1083/jcb.201803074

Figure Lengend Snippet: List of antibodies used

Article Snippet: Rabbit anti 53BP1 , Novus Biologicals (NB100-304) , IF (1:1,000).

Techniques: Western Blot

Journal: The Journal of Cell Biology

Article Title: Transcription and mRNA export machineries SAGA and TREX-2 maintain monoubiquitinated H2B balance required for DNA repair

doi: 10.1083/jcb.201803074

Figure Lengend Snippet: List of primers used

Article Snippet: Rabbit anti 53BP1 , Novus Biologicals (NB100-304) , IF (1:1,000).

Techniques: Sequencing

Journal: Cancers

Article Title: CF10 Displays Improved Synergy with Oxaliplatin in TP53 -Null and Wild-Type CRC Cells from Increased Top1cc and Replication Stress

doi: 10.3390/cancers18050882

Figure Lengend Snippet: ( A ) High throughput neutral COMET assay quantification showing increased DNA double-strand breaks 48 h post-treatment with CF10 (0.03 µM) relative to 5-FU (10 µM), and OXA (10 µM) single agents and COXA relative to FOXA. DNA tail percentages were quantified in TP53 -WT and TP53 -null HCT116 cells. ( B – D ) Western blots with quantification for TS ( B ), Chk1/pChk1 ( C ), and Chk2/pChk2 ( D ). Experiments were performed independently in triplicate per experimental point, and representative results were shown. The data are presented as a mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001, and ns = not significant. Statistical significance was assessed using a two-way analysis of variance, with p < 0.05 considered significant (GraphPad Prism 7; GraphPad Software, Inc.). The uncropped blots are shown in .

Article Snippet: Nitrocellulose membranes were blocked with 5% non-fat dry milk in TBS-T for 1 h at room temperature, then incubated with primary antibodies against p53 (Cell Signaling, Cat. No. 2524, RRID: AB_331743), p21 (Cell Signaling, Cat. No. 2947, RRID: AB_823586), β-actin (Cell Signaling, Cat. No. 4970, RRID: AB_2223172), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 2348, RRID: AB_331212), Phospho-Chk2 (Thr68) (Cell Signaling Technology Cat. No. 2197, RRID: AB_2080501), Chk1 (Cell Signaling Technology Cat# 37010, RRID: AB_3662851), Chk2 (Cell Signaling Technology Cat# 6334, RRID: AB_11178526), and Thymidylate Synthase (D5B3) (Cell Signaling Technology Cat# 9045, RRID: AB_2797693) diluted in TBS-T overnight at 4 °C.

Techniques: High Throughput Screening Assay, Neutral Comet Assay, Western Blot, Software

Journal: Nucleic Acids Research

Article Title: p53 coordinates base excision repair to prevent genomic instability

doi: 10.1093/nar/gkw015

Figure Lengend Snippet: Accumulation of SSBs negatively affects Sp1 stability and activity towards the APEX1 promoter. ( A ) Histogram displaying a ChIP analysis assessing Sp1 binding to APEX1 proximal promoter. The assay was carried out using TIG-1 cells transfected with either a control siRNA, a XRCC1 siRNA or a Sp1-targeting siRNA (as positive control for signal reduction). Results are expressed as mean fold enrichment over unspecific IgG relative to control siRNA ± SD of three independent experiments. ( B ) Representative EMSA assay measuring Sp1 binding activity to APEX1 proximal promoter. Cells were treated as described in panel A and nuclear extracts were used to assess Sp1 binding activity towards an APEX1 promoter probe. Densitometric quantification of the DNA/protein complex is reported at the bottom of the picture. ( C ) Histogram illustrating the reduction in full length APEX1 promoter activity in TIG-1 cells measured by luciferase assay upon XRCC1 knockdown. Results are expressed as mean relative promoter activity ± SD of nine independent experiments. Inset: western blotting showing a representative XRCC1 knockdown. ( D ) Representative western blotting analysis on TIG-1 cells depleted of XRCC1 and p53. Sp1 is downregulated upon XRCC1 knockdown in a p53-dependent manner. Actin was used as loading control. ( E ) Boxplot showing the distribution of Sp1 staining intensity (in arbitrary units) in p53 low versus p53 overexpressing cells. The dashed line highlights the median Sp1 intensity in p53 low cells ( N > 8000). ( F ) Representative western blotting analysis showing rescue of Sp1 levels by proteasome inhibition (MG132 used 10 μM, 6 h) in a XRCC1-depleted background. Actin was used as loading control.

Article Snippet: Four μg of either non-specific IgG (SantaCruz Biotechnology) or Sp1 antibody were added to the supernatant and rotated end-over-end overnight at 4°C.

Techniques: Activity Assay, Binding Assay, Transfection, Control, Positive Control, Luciferase, Knockdown, Western Blot, Staining, Inhibition

Journal: Nucleic Acids Research

Article Title: p53 coordinates base excision repair to prevent genomic instability

doi: 10.1093/nar/gkw015

Figure Lengend Snippet: Defective p53 activity leads to failure of the BER coordination system. ( A ) High-throughput microscopy analysis of TIG-1 cells transfected with plasmids expressing wild-type or mutant p53. Boxplots showing the distribution of APE1 staining intensity (in arbitrary units) in p53 low versus p53 overexpressing cells. Each p53 mutant is reported on top of the relevant plot. The dashed line highlights the median APE1 intensity in p53 low cells ( N > 5000). NS: not statistically significant at P < 0.05. ( B ) Representative western blotting analysis comparing WI38 and WI38 (SV40) cells upon transfection with the indicated siRNAs. Failure to downregulate Sp1 correlates with the inability to modulate APE1. Actin was used as loading control. ( C ) qPCR analysis on APE1 transcript in WI38 and WI38 (SV40) cells upon transfection with the indicated siRNAs. APE1 transcription is reduced in WI38 cells only. Note the higher transcript content in WI38 (SV40) cells. ( D ) Representative Western blotting analysis comparing WI38 and WI38 (SV40) cells upon transfection with the indicated siRNAs. Failure to modulate BER correlates with γH2AX staining in WI38 (SV40) cells; γH2AX increases further upon XRCC1 depletion. ( E ) Neutral Comet assay on WI38 and WI38 (SV40) fibroblasts shows accumulation of DSBs upon XRCC1-depletion in transformed cells only. Results are expressed as mean ± SD of three independent experiments.

Article Snippet: Four μg of either non-specific IgG (SantaCruz Biotechnology) or Sp1 antibody were added to the supernatant and rotated end-over-end overnight at 4°C.

Techniques: Activity Assay, High Throughput Screening Assay, Microscopy, Transfection, Expressing, Mutagenesis, Staining, Western Blot, Control, Neutral Comet Assay, Transformation Assay

Journal: Cancers

Article Title: CF10 Displays Improved Synergy with Oxaliplatin in TP53 -Null and Wild-Type CRC Cells from Increased Top1cc and Replication Stress

doi: 10.3390/cancers18050882

Figure Lengend Snippet: ( A ) High throughput neutral COMET assay quantification showing increased DNA double-strand breaks 48 h post-treatment with CF10 (0.03 µM) relative to 5-FU (10 µM), and OXA (10 µM) single agents and COXA relative to FOXA. DNA tail percentages were quantified in TP53 -WT and TP53 -null HCT116 cells. ( B – D ) Western blots with quantification for TS ( B ), Chk1/pChk1 ( C ), and Chk2/pChk2 ( D ). Experiments were performed independently in triplicate per experimental point, and representative results were shown. The data are presented as a mean ± SEM. * p < 0.05; ** p < 0.01; *** p < 0.001, **** p < 0.0001, and ns = not significant. Statistical significance was assessed using a two-way analysis of variance, with p < 0.05 considered significant (GraphPad Prism 7; GraphPad Software, Inc.). The uncropped blots are shown in .

Article Snippet: Nitrocellulose membranes were blocked with 5% non-fat dry milk in TBS-T for 1 h at room temperature, then incubated with primary antibodies against p53 (Cell Signaling, Cat. No. 2524, RRID: AB_331743), p21 (Cell Signaling, Cat. No. 2947, RRID: AB_823586), β-actin (Cell Signaling, Cat. No. 4970, RRID: AB_2223172), Phospho-Chk1 (Ser345) (133D3) (Cell Signaling Technology, Danvers, MA, USA, Cat. No. 2348, RRID: AB_331212), Phospho-Chk2 (Thr68) (Cell Signaling Technology Cat. No. 2197, RRID: AB_2080501), Chk1 (Cell Signaling Technology Cat# 37010, RRID: AB_3662851), Chk2 (Cell Signaling Technology Cat# 6334, RRID: AB_11178526), and Thymidylate Synthase (D5B3) (Cell Signaling Technology Cat# 9045, RRID: AB_2797693) diluted in TBS-T overnight at 4 °C.

Techniques: High Throughput Screening Assay, Neutral Comet Assay, Western Blot, Software

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Diagram of growth competition assay. mCherry-labelled RPE1 cells were mixed with unlabeled TP53 −/− RPE1 (1:1), exposed to IR and grown for 6 days. b, Relative abundance of unlabeled TP53 −/− Clone#1 measured by Intellicyte high-throughput cytometry ± SEM (n=6) is shown, normalized to the untreated (0Gy) cohort at each time point. c, Relative abundance of unlabeled TP53 −/− Clone#2 ± SEM (n=6) is shown, normalized to the untreated (0Gy) cohort at each time point. d, Representative immunofluorescence images of 53BP1 foci in cells with indicated genotypes untreated (no IR) or treated with IR (5Gy) and collected at .5, 2, and 4 h after irradiation. e, Quantification of 53BP1 foci. Data shown are mean (n=50 cells per treatment condition) ± SEM (n=3), and are consistent across two independent biological replicates. * p <0.05; ** p <0.01; by two-tailed Student’s t-test. f, Representative Neutral COMET fluorescence staining for DNA tails in cells with indicated genotypes treated with or without 5Gy IR. For irradiated cells, 2 timepoints are shown: immediately after and 4 hours post IR. COMET tails and heads are denoted by OpenComet software analysis. g, Quantification of DNA DSBs via Neutral COMET assay reported as tail DNA percent at 0 and 4 hours post IR in RPE1 and two TP53 −/− RPE1 cell lines. Data shown are mean (n= 50-150 cells per treatment condition) ± SEM, and are consistent across three independent biological replicates. * p <0.05; ** p <0.01; **** p <0.0001 by two-tailed Student’s t-test.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Competitive Binding Assay, High Throughput Screening Assay, Cytometry, Immunofluorescence, Irradiation, Two Tailed Test, Fluorescence, Staining, Software, Neutral Comet Assay

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Live cell imaging procedure. Cells transfected with 10 nM si-control or si- TP53 for 48 h prior to imaging. 18 h into imaging, cells are treated with NCS (100 nM), DNA-PKi (.5 uM) or both and imaged for 72 total hours. b, RPE1 cell expressing the PCNA-mCherry and 53BP1-mVenus reporters. Cell cycle phases delineated by PCNA foci and DNA DSBs are marked by 53BP1 foci. c, RT-qPCR for TP53 mRNA levels (left) and CDKN1A mRNA levels (right) in si-control treated vs. si- TP53 treated cells. To induce CDKN1A expression, cells irradiated at 5Gy and mRNA harvested 3 hrs post IR. d, Heatmap of 53BP1 foci tracings for single cells tracked from birth to mitosis or end of imaging. For si-control (n = 30 cells) and si- TP53 treated RPE1 (n = 60 cells) treated with NCS 100 ng/ml. For visualization, cells are aligned to 10 frames prior to drug addition (black arrow). e, Heatmap of 53BP1 foci tracings for si-control (n = 25 cells) and si- TP53 treated RPE1 cells (n = 55 cells) treated with 100 ng/ml NCS + 0.5 uM DNA-PKi. f, Peak 53BP1 foci counts for cells treated with 100 ng/ml NCS or NCS+0.5 uM DNA-PKi. Significance determined using two-tailed t-test. g, Area under the curve (AUC) analysis of 53BP1 burden showing integral DNA damage for cells treated with NCS vs. NCS and DNA-PKi. Cells are segregated into two groups: cells exposed to drug in G1 vs. S phase (n = 25-30 G1or S cells for si- TP53 cohort, n = 10-15 G1 or S cells for si-control cohort). Significance determined by two-tailed t-test. **** p <0.0001, *** p <0.001, n.s. = non-significant. h, 53BP1 foci burden in G1 vs. S phase p53-deficient RPE1 upon exposure to NCS and DNA-PKi. Dashed line = S phase onset, blue line = mean 53BP1 foci burden for all cells in G1 with NCS and DNA-PKi addition, orange line = mean foci value for cells in G1 with NCS treatment alone, (n = 30 cells for each condition).

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Live Cell Imaging, Transfection, Control, Imaging, Expressing, Quantitative RT-PCR, Irradiation, Two Tailed Test

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Normal Mitosis: RPE1 Cell cycle representative of normal mitosis, with NCS treatment only. For all cells in this figure both the PCNA and the 53BP1 channels are shown as two individual movies. b, Transient G2 Delay: RPE1 cell cycle representative of a transient cell cycle delay in G2 (length of G2 is significantly prolonged in comparison to untreated cells). This cell was treated with NCS and DNA-PKi. c, G1 Arrest: RPE1 cell cycle representative of a permanent G1 arrest. This is a p53 proficient cell treated with NCS and DNA-PKi.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Comparison

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Cell cycle outcome analyses for si-control treated RPE1, dashed white line indicates drug addition, each row is an individual cell (n = 60 cells for NCS and n=60 cells for NCS+DNA-PKi treatment). Colored bars indicate different phases of the cell cycle, legend shown with no treatment control for comparison. Cells with red bars at the end of mitosis indicate terminal cell cycle event (mitotic catastrophe or apoptosis). Event frequency is reported as a percentage on the right. Cells exposed in G1 vs. S cells are treated as separate cohorts. Fisher’s exact test was performed between −/+ DNA-PKi cohorts using 2 outcome groups (viable, vs. non-viable (arrested cells + terminal outcomes). **** p <0.0001, n.s. =non-significant b, Cell cycle outcome analyses for si- TP53 treated RPE1, dashed line indicates drug addition, each row is an individual cell (n = 60 cells for NCS and n=60 cells for NCS+DNA-PKi treatment). c, AUC analysis of 53BP1 damage burden in viable vs. non-viable p53-deficient cells that were treated with NCS and DNA-PKi. Statistical significance was calculated using a Mann-Whitney test comparing ranks. **** p <0.0001 d, Dynamics of 53BP1 foci burden p53-deficient RPE1 segregated by mitotic viability. The red line corresponds to mean 53BP1 foci burden for all p53-deficient cells treated with NCS and DNA-PKi that undergo catastrophic mitoses, black line indicates mean foci value for p53-deficient cells with NCS and DNA-PKi treatment that are viable post mitosis, (n = 20 viable cells and n = 33 non-viable cells).

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Control, Comparison, MANN-WHITNEY

Journal: bioRxiv

Article Title: Hyperactive end joining repair mediates resistance to DNA damaging therapy in p53-deficient cells

doi: 10.1101/2020.04.01.021253

Figure Lengend Snippet: a, Time stamped image sequence of apoptotic cell (PCNA channel shown). Cells that experienced nuclear degradation during cell cycle prior to mitosis were categorized as “apoptotic cells.” In this sequence a cell in G2 experiences cell death at 27 hours post birth, with indication of mitotic attempt, with nuclear envelope collapse or presence of any daughter cells. b, Time stamped image sequence of cell that experienced mitotic catastrophe (PCNA channel shown). Cell undergoes nuclear envelope collapse (24:10), and attempts mitosis, in subsequent images fragmentation of nucleus is clearly visible with no viable daughter cells present. Cell non-viability during mitosis was defined as mitotic catastrophe. c, Integral DNA damage burden for p53-deficient cells treated with NCS (100 ng/ml) and DNA-PKi (.5 uM) are calculated and segregated by viable (black) vs. non-viable outcomes (red). Legend indicates which phase of cell cycle the cells are in during drug exposure, followed by the phase for which the burden is calculated. Ex: G1 cells G1 burden = cells in G1 during drug exposure and total damage burden in G1. Area under the curve (AUC) analysis was performed by plotting 53BP1 foci counts over time for each cell and integrating burden over time. Statistical significance was determined using two-tailed Student’s t-test.

Article Snippet: The primary antibodies used were: γH2AX (1:500, Trevigen, 4418-APC-100), and 53BP1 (1:500 for immunofluorescence, Bethyl, A300-272A).

Techniques: Sequencing, Two Tailed Test