Journal: Oncotarget

Article Title: DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response

doi:

Figure Lengend Snippet: (A and B) BMH-21-caused nucleolar stress is independent of ATM pathway activation. A375 cells were pretreated with ATM inhibitor (ATMi) KU55933 (10 μM) for 30 min as indicated, followed by treatment with BMH-21 (1 μM) or IR (2 Gy) and incubation for 3 h. Cells were stained for (A) S1891-phosphorylated ATM (PATM, green ) or (B) NCL ( green ) and counterstained for DNA ( blue ). (C) Parent DLD and DLD cells with ATR-knock in mutation (DLD Seckel cells) were treated with BMH-21 for 6 h followed by staining for NPM ( green ). Merged images with DNA ( blue ) are shown. (D) Inhibition of DDR pathways does not affect BMH-21-mediated RPA194 degradation. A375 cells were pretreated for 30 min with the following: KU55933 (10 μM), caffeine (2 mM), wortmannin (10 μM), NU7441 (5 μM) followed by addition of BMH-21 (1 μM) and incubation for 2 h. Cells were stained for RPA194 ( green ), UBF ( red ) and counterstained for DNA ( blue ) Arrowheads , nucleolar caps. (E) A375 cells were pretreated with KU55933 (10 μM) for 1 h as indicated, followed by IR (2 Gy) and incubation for the indicated times. BMH-21 (1 μM) was added for the final 3 h as indicated. Cell lysates were analyzed by western blotting for RPA194 and GAPDH was used as a loading control. (F) A375 cells were pretreated with NU7441 (10 μM) for 1 h as indicated, followed by addition of ActD (50 ng/ml) or BMH-21 (1 μM) and incubation for 3 h. Cell lysates were analyzed by western blotting for RPA194, NCL and GAPDH was used as a loading control. Scale bars, 10 μm.

Article Snippet: Other reagents were KU55933 and caffeine (Calbiochem), ActD, camptothecin, wortmannin (Sigma) and NU7441 (Santa Cruz Biotechnology).

Techniques: Activation Assay, Incubation, Staining, Knock-In, Mutagenesis, Inhibition, Western Blot, Control

Journal: Oncotarget

Article Title: DNA intercalator BMH-21 inhibits RNA polymerase I independent of DNA damage response

doi:

Figure Lengend Snippet: U2OS cells were treated with LI-216 (10 μM) for 3 h in the presence or absence of NU7441 (10 μM). Cells were fixed and stained for (A) PATM, (B) γH2AX, (C) PKAP1, (D) PDNA-PK and counterstained for DNA. Scale bars, 10 μm.

Article Snippet: Other reagents were KU55933 and caffeine (Calbiochem), ActD, camptothecin, wortmannin (Sigma) and NU7441 (Santa Cruz Biotechnology).

Techniques: Staining

Journal: bioRxiv

Article Title: Aberrant inheritance of extrachromosomal DNA amplifications promotes cancer evolution

doi: 10.1101/2025.09.19.677276

Figure Lengend Snippet: a, Correlation between ecDNA amount (ecDNA area, x axis) and untethered ecDNA (untethered area, y axis) as determined using DNA-FISH (see methods for computational details). Pearson correlation r=0.4746 one-tailed (n=3) shows a significant positive correlation between the overall amount of ecDNA and the amount of untethered ecDNA in mitotic cells. b, Quantification of ecDNA tethering during early and late mitosis in HeLa clone 1 cells with an average of 52 and 162 ecDNA. Two-way ANOVA followed by Šídák’s multiple comparisons test, mean ± SEM (n=3). c, Representative DNA-FISH images of ecDNA tethering in HeLa clone 1 cells with an average of 52 ecDNA. d, Quantification of FGFR2 + and MYC + ecDNA tethering during early and late mitosis in SNU-16 cells. Linear mixed model fit, mean ± SEM (n=4). e, Representative DNA-FISH images of daughter HeLa clone 1 cells in interphase showing even and uneven segregation of DHFR + ecDNA (red). Numbers denote ecDNA quantification (see methods for details). f, Quantification of ecDNA inheritance in HeLa clone 1 cells containing an average of 29, 52, and 362 ecDNA (pink). Y-axis presents the percent of ecDNA in the daughter cell inheriting the lower amount of ecDNA (“unlucky”). Simulations of ecDNA inheritance (blue) assuming random binomial distribution (p=0.5) for each ecDNA amount are presented. Comparison performed using pairwise nonparametric test. Mean±SEM (n=3) are presented. See method section for more details. g, Quantification of MYCN+ ecDNA inheritance in CHP-212 cells (n=3). MYCN + HSR+ SK-N-BE(2) cells were used as control (n=3). Welch’s t-test two-tailed; Mean±SEM. h, Simulation of the number of population doublings required for HeLa cells resistant to 1600nM MTX (containing an average of 74±80 ecDNA) to adapt to 3200nM MTX (reaching an average of 162±106 ecDNA). Comparison of even (p=0.5) and uneven (p=0.4, based on experimental data from ) is presented. Two-tailed student t-test. Mean±SD. i, Representative DNA-FISH images of HeLa clone 1 cells containing an average of 362 ecDNA treated with 500 nM JQ1+ or DMSO (control) for 24h. Note the reduced clustering on mitotic chromosomes in JQ1 treated cells. j, Quantification of ecDNA aggregate sizes tethered to mitotic chromosomes using DNA-FISH (as shown in panel i) in HeLa clone 1 containing an average of 362 ecDNA cell with or without JQ1 treatment. Size of larger (over 100 pixels) aggregates of tethered ecDNA is presented. Welch’s t-test (one tailed); mean+SEM (n=3). k, Quantification of ecDNA inheritance in CHP-212 cells treated with or without 500 nM JQ1 for 24h. Unpaired one-tailed student t-test (n=3). Mean±SEM.

Article Snippet: Acute drug treatments: HeLa clone 1 cells were seeded on coverslips and 24 h afterwards had their medium change with one of the following drugs: Reversine (500 nM, 24h), JQ1 (500nM, 24h), Nu7441(Selleckchem; s2638, 10 μM, 48 h), Etoposide (Sigma, E1383; 5μM for two hours and release for 24 hours).

Techniques: One-tailed Test, Comparison, Control, Two Tailed Test

Journal: bioRxiv

Article Title: Aberrant inheritance of extrachromosomal DNA amplifications promotes cancer evolution

doi: 10.1101/2025.09.19.677276

Figure Lengend Snippet: a, Snapshots from Supplementary Video #1 showing a dividing H2B-mCherry+ HeLa clone 1 containing 362 ecDNA cell (from a population with an average of 362 ecDNA). Note the detached ecDNA during mitosis that remain in the cytoplasm following division. b, Description of the four micronucleus types and their origin as identified in this study. See main text for more details. c, Quantification of micronucleus types in HeLa cells without amplification, with HSR, and with increasing amounts of ecDNA. One-way ANOVA followed by Tukey’s multiple comparisons, mean ± SEM (n=3). d, Quantification of micronucleus types in neuroblastoma cell lines. One-way ANOVA followed by Tukey’s multiple comparisons test, mean ± SEM (n=3). SH-SY5Y – no amplification, SK-N-BE(2) – MYCN + HSR, CHP-212 – MYCN + ecDNA. e, Quantification of micronucleus types in six neuroblastoma patients (n=3 ecDNA-, n=3 ecDNA + ). Two-way ANOVA followed by Šídák’s multiple comparisons test, mean ± SEM. f, Representative DNA-FISH image of MYCN + ecDNA + neuroblastoma patient sample. Inserts show examples of DAPI+ (1), hitchhiker (2), hub (3), and singlet (4) micronucleus types. g-h, Quantification of ecDNA+ micronucleus sizes in HeLa clone 1 cells with an increased number of ecDNA (g) and in CHP-212 and COLO320DM cells (h). One-way ANOVA followed by Tukey’s multiple comparisons test, mean ± SEM (n=3). i, Representative DNA-FISH image of HeLa clone 1 cells with 362 ecDNA showing different sizes of ecDNA+ micronuclei. White numbers denote micronucleus size (see methods for details). j,l, Quantification of micronucleus types in HeLa clone 1 containing 362 ecDNA after 500 nM JQ1 treatment for 24h (j) or 500nM reversine treatment for 24h (l) treatment. Two-way ANOVA followed by Šídák’s multiple comparisons test, mean ± SEM (n=3). k,m, Quantification of ecDNA+ micronucleus sizes in HeLa clone 1 containing 362 ecDNA after 500 nM JQ1 treatment for 24h (k) or 500nM reversine treatment for 24h (m). Welch’s t-test two-tailed, mean ± SEM (n=3). n, Representative DNA-FISH image of HeLa clone 1 containing 52 ecDNA treated with 500 nM 24h reversine. o,q, Quantification of lamin B1+ micronuclei in a HeLa clone 1 containing 52 ecDNA (o) and SNU-16 cells (q). Two-way ANOVA followed by Tukey’s multiple comparisons test, mean ± SEM (n=3). p, Representative IF-FISH image of lamin B1 negative singlet and hub-type mirconuclei in HeLa clone 1 containing 52 ecDNA. r, Quantification of cGAS+ micronuclei in HeLa clone 1 containing 52 ecDNA. Data are shown as mean ± SEM (n=3). s, Representative IF-FISH image of cGAS+ singlet and hub-type micronuclei. t, Ingenuity pathway analysis showing top upstream regulators (top 10 and cGAS which was number 23) when comparing HeLa clones 1-3 at 10,000 nM MTX resistance to control HeLa S3 cells (n=4). Blue highlight the cGAS and STING pathways. Y axis shows p-value for each pathway.

Article Snippet: Acute drug treatments: HeLa clone 1 cells were seeded on coverslips and 24 h afterwards had their medium change with one of the following drugs: Reversine (500 nM, 24h), JQ1 (500nM, 24h), Nu7441(Selleckchem; s2638, 10 μM, 48 h), Etoposide (Sigma, E1383; 5μM for two hours and release for 24 hours).

Techniques: Amplification, Two Tailed Test, Clone Assay, Control

Journal: bioRxiv

Article Title: Aberrant inheritance of extrachromosomal DNA amplifications promotes cancer evolution

doi: 10.1101/2025.09.19.677276

Figure Lengend Snippet: a. Quantification of micronuclei percent (relative to primary nuclei) in a HeLa clone 1 containing 52 ecDNA following 100 nM bafilomycin A1 treatment for 6 to 24 hours. Two-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean ± SEM (n=3). *** p=0.0001, **** p<0.0001 b. Representative images showing of ecDNA+ micronuclei in HeLa clone 1 containing 52 ecDNA cells treated with 100 nM Bafilomycin A1 for 6 to 24 hours. c. Quantification of micronuclei percent (relative to primary nuclei) following ATG3 siRNA-mediated knockdown (72 hours) in HeLa clone 1 containing 52 ecDNA (n=2) and SNU-16 (n=1) cells. Two-way ANOVA followed by Tukey’s multiple comparisons test. Data are presented as mean ± SEM. d. Representative images showing increased DHFR⁺ ecDNA-containing micronuclei in the cytosol upon ATG3 knockdown of HeLa clone 1 containing 52 ecDNA. e. Quantification of micronuclei percent (relative to primary nuclei) following FIP200 siRNA-mediated knockdown (72 hours) in HeLa clone 1 containing 52 ecDNA. Two-way ANOVA followed by Tukey’s multiple comparisons test (n=3). f, Illustration of experimental set-up performed in panel g. HeLa clone 3 cells were treated with combinations of 200 nM reversine and 800 nM MTX for 1 week and metaphase spreads were prepared for DNA-FISH. g, Percent of colocalization of DHFR and ARHGEF28 genes per cell in HeLa clone 3 treated with 200 nM ± reversine and 800 nM ±MTX (see panel f) for 1 week. One-way ANOVA with Dunnett’s multiple comparisons test (n=3). Data are presented as mean ± SEM. * p=0.0316, ** p=0.0012, *** p=0.0001 h, Graphical summary of study observations. ecDNA aggregation promotes uneven mitotic distribution leading to rapid accumulation and heterogeneity in the progeny. Cytosolic mis-segregated ecDNA encapsulate in different micronucleus types, driving structural evolution through potential chromothriptic rearrangements or autophagic degradation.

Article Snippet: Acute drug treatments: HeLa clone 1 cells were seeded on coverslips and 24 h afterwards had their medium change with one of the following drugs: Reversine (500 nM, 24h), JQ1 (500nM, 24h), Nu7441(Selleckchem; s2638, 10 μM, 48 h), Etoposide (Sigma, E1383; 5μM for two hours and release for 24 hours).

Techniques: Knockdown

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: Scientific Reports

Article Title: Histone H4 expression is cooperatively maintained by IKKβ and Akt1 which attenuates cisplatin-induced apoptosis through the DNA-PK/RIP1/IAPs signaling cascade

doi: 10.1038/srep41715

Figure Lengend Snippet: ( a,b ) The cells were pre-treated with DNA-PK inhibitor NU7026 (10 μM) for 1 h or left untreated and treated with cisplatin (7.5 μM) treatment for another 72 h. Cell death was measured by LDH releasing assay. Columns, mean of three experiments; bars, SD. ** p < 0.01. ( c ) The cells were treated with cisplatin (7.5 μM) for 30 min. The expression of the indicated proteins was detected by Western blot. GAPDH was detected as a loading control. ( d,e ) The cells were pre-treated with NU7026 (10 μM) for 1 h or left untreated followed by cisplatin (7.5 μM) treatment for another 2 h. The expression of the indicated proteins was detected by Western blot. GAPDH and Lamin B1 were detected as a loading control.

Article Snippet: The pan-caspase inhibitor zVAD and DNA-PK inhibitor NU7026 were the products of Merck Millipore (Billerica, MA, USA).

Techniques: Expressing, Western Blot, Control

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