Journal: eLife

Article Title: Endogenous tagging using split mNeonGreen in human iPSCs for live imaging studies

doi: 10.7554/eLife.92819

Figure Lengend Snippet:

Article Snippet: Chemical compound, drug , NU7441 , Tocris Bioscience , 3712 , NHEJ inhibitor.

Techniques: Recombinant, Sequencing, Ligation, Digital PCR, Software, Polymer, Imaging

Journal: Disease Models & Mechanisms

Article Title: CRISPR/Cas9-mediated homology-directed repair by ssODNs in zebrafish induces complex mutational patterns resulting from genomic integration of repair-template fragments

doi: 10.1242/dmm.035352

Figure Lengend Snippet: Influence of chemical compound administration through injection on HDR efficiency. Injection mixes containing the NT 120 S repair template were complemented with chemical compounds that either inhibit specific components of the NHEJ pathway, including SCR7, NU7441 and KU0060648, or that were shown to stimulate the HDR pathway, including RS1 and L755507. Five independent experiments were carried out and average total HDR rates are shown, split into two categories: perfect repair % (plain bars) and erroneous repair % (dashed bars). Error bars represent the s.e.m. for five independent biological replicates each consisting of a pooled sample of 20 embryos. Repair rates depicted in this graph are listed in Table S4 . Statistical tests performed: independent samples t -test for normal distributed groups and the non-parametric independent samples Mann–Whitney U -test for non-normal distributed groups.

Article Snippet: The following chemical compounds were dissolved in DMSO and either supplemented to the injection mix ( Table S9 ) or to the compound screening medium ( Table S10 ): SCR7 (Xcessbio Biosciences Inc., cat. no. M60082-2s), KU0060648 (APExBIO, cat. no. A1769), NU7441 (Santa Cruz Biotechnology, cat. no. sc-208107), L755507 (Santa Cruz Biotechnology, cat. no. sc-204045), RS1 (Santa Cruz Biotechnology, cat. no. sc-222240).

Techniques: Injection, MANN-WHITNEY

Journal: Frontiers in Immunology

Article Title: DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response

doi: 10.3389/fimmu.2022.1042463

Figure Lengend Snippet: DNA-PKcs is critical for control of ZIKV infection. Virus replication measured by plaque assay, expressed as plaque-forming units per mL (PFU/mL), on (A) A549 WT and A549 PRKDC-/- or (B) RPE WT and RPE PRKDC-/- cells infected with ZIKV at indicated m.o.i. and time. RT-qPCR analysis to measure ZIKV RNA in (C) A549 WT and A549 PRKDC-/- or (D) RPE WT and RPE PRKDC-/- cells infected at indicated m.o.i. and time. (E) A549 WT and A549 PRKDC-/- or (F) RPE WT and RPE PRKDC-/- cells infected with 50 PFU of ZIKV at 48 hours in semi-solid medium, then ZIKV-E protein (green) was stained for immunofluorescence analysis, and the relative area of infection percentage was measured using the ImageJ software. The cell nuclei were stained with DAPI (blue). (G) Percentage of ZIKV-infected A549 WT and A549 PRKDC-/- cells at indicated m.o.i. and time, analyzed by flow cytometry. (H) Viability analysis by MTT assay of ZIKV-infected A549 WT and A549 PRKDC-/- cells relative to uninfected cells (mock) at indicated m.o.i. and time. (I) A549 WT and A549 PRKDC-/- were pretreated with NU7441 (0.5 and 1 µM) at 24 hours followed by ZIKV infection (m.o.i. 1) at 24 hours. We used two-way ANOVA with Sidak’s correction in (A–D, G, H) , and unpaired two-tailed Student’s t-test was used in (E, F) . * p<0.05, n = 3, error bars ± SEM.

Article Snippet: Cells were treated with 0.5 or 1 μM of NU7441 (BioGems, 5039598), 100 ng/mL human TNF (Peprotech, 300-01A), and 3 μM etoposide (Sigma, E1383).

Techniques: Control, Infection, Virus, Plaque Assay, Quantitative RT-PCR, Staining, Immunofluorescence, Software, Flow Cytometry, MTT Assay, Two Tailed Test

Journal: Frontiers in Immunology

Article Title: DNA-PKcs restricts Zika virus spreading and is required for effective antiviral response

doi: 10.3389/fimmu.2022.1042463

Figure Lengend Snippet: ZIKV infection does not induce DSB in A549 cells. A549 WT , A549 PRKDC-/- and 1 µM NU7441 pre-treated A549 WT infected with ZIKV (m.o.i. 1) at 24 hours. Stimulation with 3 µM etoposide for 12 hours was used as a DSB positive control. (A) Immunofluorescence to analyze γH2AX (green) in the ZIKV-infected cells (red, ZIKV-E protein). The cell nuclei were stained with DAPI (blue). (B) Percentage of γH2AX foci per cell showed in (A) . *Compared with WT cells; #Compared with mock. We used two-way ANOVA with Sidak’s correction. * or # p<0.05, n = 3, error bars ± SEM.

Article Snippet: Cells were treated with 0.5 or 1 μM of NU7441 (BioGems, 5039598), 100 ng/mL human TNF (Peprotech, 300-01A), and 3 μM etoposide (Sigma, E1383).

Techniques: Infection, Positive Control, Immunofluorescence, Staining

Journal: Molecular Cell

Article Title: Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

doi: 10.1016/j.molcel.2018.04.016

Figure Lengend Snippet: Multiple Repair Pathways Active at One Locus (A and B) The spectrum of indels and their frequencies at the LBR2 locus at time point t = 60 hr in cells cultured without (A) or with (B) 1 μM NU7441. A representative experiment is shown. Light-blue bar, wild-type sequence; dark-blue bars, indels. (C) Frequencies of −7 and +1 indels in the presence (black, n = 7) and absence (gray, n = 4) of NU7441. All series are normalized to the total indel fraction. Asterisks indicate p values according to Student’s t test: ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005. (D) The −7 deletions consist of two types; red nucleotides mark the deleted DNA. Shaded nucleotides show possible models for microhomology-mediated repair. Percentages indicate the proportion of observed −7 sequence reads. (E) TIDE analysis of +1 insertion and −7 deletion indels after exposure of the cells to 10 Gy of IR at the time of Cas9 induction. Asterisks indicate p values according to Student’s t test: ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005.

Article Snippet: DNA-PKcs inhibitor NU7441 (Cayman) (final concentration 1 μM) or DMSO (control) was added to K562#17 at the same time when the cells were supplemented with Shield-1 to induce DD-Cas9.

Techniques: Cell Culture, Sequencing

Journal: Molecular Cell

Article Title: Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

doi: 10.1016/j.molcel.2018.04.016

Figure Lengend Snippet: MMEJ Has Slower Repair Kinetics Than C-NHEJ (A) Kinetic model of Cas9-induced DSB repair assuming that each type of indel is generated with a specific repair rate. (B) +1 insertion and −7 deletion accumulation in a representative time series. Dashed lines show a Gompertz sigmoid fit. (C) Same measurements as in (B) with the multi-indel ODE model fit (solid lines) for the +1 (top) and −7 (bottom) indels. (D) Cartoon representation of hypothesized time dependency of the k +1 and k −7 rates added to the fitted model in (E). (E) Time-dependent ODE model fit of the +1 insertion (top) and −7 deletion (bottom) as proposed in (D). (F) Rate constants without (black) and with (gray) NU7441. The total mutagenic repair rate is represented by k m ; rate constant for the +1 insertion is k +1 and rate constants for the −7 deletion are k −7,t = 0hr and k −7,t = 60hr at the start and end of the time series, respectively. n indicates the number of time series. Asterisks indicate p values according to Wilcoxon test: ∗ p < 0.05; ∗∗ p < 0.005; ∗∗∗ p < 0.0005. (G) Broken fraction measurements in presence of NU7441 of 2 independent time series, similar to B and 3C.

Article Snippet: DNA-PKcs inhibitor NU7441 (Cayman) (final concentration 1 μM) or DMSO (control) was added to K562#17 at the same time when the cells were supplemented with Shield-1 to induce DD-Cas9.

Techniques: Generated

Journal: Molecular Cell

Article Title: Kinetics and Fidelity of the Repair of Cas9-Induced Double-Strand DNA Breaks

doi: 10.1016/j.molcel.2018.04.016

Figure Lengend Snippet:

Article Snippet: DNA-PKcs inhibitor NU7441 (Cayman) (final concentration 1 μM) or DMSO (control) was added to K562#17 at the same time when the cells were supplemented with Shield-1 to induce DD-Cas9.

Techniques: Virus, Recombinant, Viability Assay, Bicinchoninic Acid Protein Assay, Imaging, Sequencing, Software

Journal: The Journal of Biological Chemistry

Article Title: Deubiquitinase USP2 stabilizes the MRE11–RAD50–NBS1 complex at DNA double-strand break sites by counteracting the ubiquitination of NBS1

doi: 10.1016/j.jbc.2022.102752

Figure Lengend Snippet: USP2 is recruited to DSB sites in a manner dependent on ATM, PARP, and RECQL4. A and B , recruitment of EGFP-USP2 to laser microirradiation sites was analyzed in U2OS cells treated with inhibitors (20 μM) of ATM (KU55933), DNA-PK (NU7441), PARP (olaparib), or PARG (PDD00017273) 1 h before microirradiation. Data in graphs are means ± SEM.; n = 20. C , recruitment of USP2 to microirradiation sites in mock- (siGL) or RECQL4-depleted (siR4) U2OS cells. Data in graphs are means ± SEM; n = 20. D , RECQL4-depleted U2OS cells were transfected with WT or Walker B mutant (WB) RECQL4 plasmids and used for analyzing USP2 binding to microirradiation sites. Data in graphs are means ± SEM; n = 20. E and F , interaction between USP2 and RECQL4 in U2OS cells. U2OS cells transfected with FLAG-RECQL4 and HA-USP2 plasmids were used for IP with anti-HA antibodies ( upper panels ) or anti-FLAG-M2-agarose beads ( lower panels ). Cells treated with NCS ( E ) or NCS and ATM inhibitor (KU55933, 20 μM) ( F ) were used for IP. Anti-FLAG (for RECQL4) and anti-HA (for USP2) antibodies were used for Western blotting. Lanes: In, 10% of input for IP; C, control IP with nonspecific IgG. DSB, double-strand break; IP, immunoprecipitation.

Article Snippet: The DNA-PK inhibitor, NU7441 (Axon 1463), was from Axon Medchem (Axon2678).

Techniques: Transfection, Mutagenesis, Binding Assay, Western Blot, Control, Immunoprecipitation