Journal: The Journal of Immunology Author Choice

Article Title: NR4A nuclear receptor expression in human macrophages mediates apoptosis and controls Mycobacterium tuberculosis growth

doi: 10.1093/jimmun/vkaf252

Figure Lengend Snippet: Macrophage apoptosis in response to pharmacologic treatment is reduced by NR4A knockdown. (A–C) AML cells were transfected with 15 μM of either scrambled siRNA or siRNA targeting NR4A1, NR4A2, or NR4A3. After 24 h, they were left untreated or treated with 1 μM staurosporine for 8 h, 10 μM actinomycin D for 24 h, or 100 μM etoposide for 24 h. Caspase-3 and caspase-7 cleavage activity was measured by luminescent assay. (D) Representative Western blot showing total and cleaved PARP1 protein expression in AML cells following treatment with staurosporine. (E) Quantified Western blot data are shown from (D). P values are indicated as asterisks above the plots as obtained from one-way ANOVA (* P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001) from n = 3 (A–C) or n = 4 (D, E) unique donors. Data are shown as mean ± SEM.

Article Snippet: Transfected AML cells in 96-well plates were treated with InSolution Staurosporine (Thermo Fisher Scientific), actinomycin D (Tocris Bioscience), or etoposide (Thermo Fisher Scientific) to induce cell death, or infected with M.tb at MOI of 5 for 24 to 72 h. Cell death was assayed in triplicate with the CellTiter Glo Assay (Promega) following the manufacturer’s instructions.

Techniques: Knockdown, Transfection, Activity Assay, Luminescence Assay, Western Blot, Expressing

Journal: International Journal of Molecular Sciences

Article Title: Therapeutic Potential for Regulation of the Nuclear Factor Kappa-B Transcription Factor p65 to Prevent Cellular Senescence and Activation of Pro-Inflammatory in Mesenchymal Stem Cells

doi: 10.3390/ijms22073367

Figure Lengend Snippet: List of drugs.

Article Snippet: Cellular senescence was induced in proliferative UC-MSCs using two models: (1) DDIS: the UC-MSCs were cultured with DMEM supplemented with 10% ( v/v ) FBS, 1% ( v/v ) penicillin, 1% ( v/v ) streptomycin (all from Sigma-Aldrich, Madrid, Spain) and 1-μM Etoposide (MedChemexpress, New Jersey, USA) ( ) for two days.

Techniques: Concentration Assay

Journal: bioRxiv

Article Title: Endocytic control of cell-autonomous and non-cell-autonomous functions of p53

doi: 10.1101/2025.08.16.670648

Figure Lengend Snippet: A. Ctrl-KO and SNX9-KO MCF10A cells were treated with etoposide at 25 and 50 μM (indicated by triangles) and analyzed by IB for total and phosphorylated (p53 ser15 ) p53, and the p53 target genes MDM2 and CDKN1A. Vinculin (VCL), loading control. B . RT-qPCR analysis of MDM2 and CDKN1A expression in the samples described in “A”. Data are from three independent experiments and expressed as mean ± SD. *, p<0.05; **, p<0.01. C. 3D reconstruction of MCF10A-p53-KO recipient cells treated for 8 h with EVs purified from the conditioned medium of HEK-293 cells transfected with p53-HA. Recipient cells were treated with etoposide (50 μM for 8 h) and analyzed by IF using an anti-p53 antibody (green) and DAPI (blue). Bar, 20 μm. D. MCF10A-p53-KO recipient cells were treated for 8 h with etoposide (50 μM) and EVs purified from HEK-293 WT (EV) or HEK-293 p53-HA (EV p53-HA) cells as indicated. After treatment cells were harvested and analyzed by RT-qPCR for CDKN1A levels. Data are from three independent experiments and expressed as mean ± SD. * p<0.05, ** p<0.01 and *** p<0.001 vs . same condition in cells not treated with EVs (only the most relevant statistical comparisons are shown). E. Scheme of the co-culture experiment shown in in panel F. a) MCF10A-p53-KO-H2B-Cherry cells were plated onto coverslips. b) After 24 h, coverslips were harvested and seeded (c) in plates in which the indicated cell lines had been previously seeded. Cells were then treated with etoposide (50 μM) or mock-treatment for 8 h. d) Coverslips were harvested and analyzed for purity of H2B-Cherry labeled cells (Fig. S10D) and for the levels of CDKN1A mRNA (panel F). Details are in Materials and Methods. F . RT-qPCR analysis of CDKN1A levels in harvested H2B-Cherry labeled MCF10A-p53-KO cells, co-cultured as described in panel “ E ”. Data are from three independent experiments and expressed as mean ± SD. **, p<0.01; n.s., not significant (only the most relevant statistical comparisons are shown). G . SAOS2 cells were treated with EVs purified from the indicated MCF10A cell lines (see experimental scheme in Fig. S10F), and cell viability/growth was assessed indirectly by quantifying intracellular ATP levels using a luminescence-based assay. Data are from ten samples/condition from two independent experiments and expressed as mean ± SD. One-way ANOVA test: *, and ***, p < 0.05 and < 0.001, respectively, vs . SAOS2 treated with EVs derived from MCF10A-p53-KO cells.

Article Snippet: Chemicals were: FLAG peptide, cat. F3290 (Merck Life Science); HA peptide, cat. 11666975001 (Merck Life Science); NUMB peptide corresponding to amino acids 537-551 of hNUMB (Genscript); Etoposide, cat. E1383 (Merck Life Science); Trehalose Dihydrate, cat. T9531 (Merck Life Science); Chloroquine, cat. C6628 (Merck Life Science); Ionomycin, cat. I0634 (Merck Life Science); Proteinase K, P4850 (Merck Life Science); MG132, cat. 474790 (Merck Life Science); U73122, cat. 6756 (Merck Life Science); GW4869, cat. S7609 (Selleck Chemicals); brain PI(4,5)P2, cat. 840046P; brain PI(4)P, cat. 840045P; brain PS, cat. 840032C; brain PC, cat. 840053C (all from Merck Life Science).

Techniques: Control, Quantitative RT-PCR, Expressing, Purification, Transfection, Co-Culture Assay, Labeling, Cell Culture, Luminescence Assay, Derivative Assay

Journal: Plants

Article Title: The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

doi: 10.3390/plants10122568

Figure Lengend Snippet: Defects in DSB repair and mitosis in topbp1 mutant. ( a ) Diagram with the mean number of leaves per seedling in the WT and topbp1 mutant grown just in MS medium or supplemented with cisplatin (30 μM) or cisplatin + etoposide (5 μM). Data collected from three independent experiments ( n = 100 per treatment and day). ( b ) Mitotic anaphases of the WT and topbp1 . Statistical differences between the WT and topbp1 for each treatment analysed by Mann–Whitney test, *** p < 0.001; ns = not significant. Comparisons among treatments within the same genetic background are shown in . Scale bar is 5 μm.

Article Snippet: Etoposide (Tocris Bioscience, Bristol, UK) at 5 μM was also used in conjunction with the DSB repair assessment.

Techniques: Mutagenesis, MANN-WHITNEY

Journal: Plants

Article Title: The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

doi: 10.3390/plants10122568

Figure Lengend Snippet: Results of the pairwise comparison of the mean number of leaves per seedling untreated (MS), treated with cisplatin, and treated with cisplatin + etoposide (Cis + Etop) by the Kruskal–Wallis test followed by Dunn’s post-hoc test in the WT and topbp1 at days 7, 12, and 16.

Article Snippet: Etoposide (Tocris Bioscience, Bristol, UK) at 5 μM was also used in conjunction with the DSB repair assessment.

Techniques: Comparison

Journal: Plants

Article Title: The Role of DNA Topoisomerase Binding Protein 1 (TopBP1) in Genome Stability in Arabidopsis

doi: 10.3390/plants10122568

Figure Lengend Snippet: Meiotic stages of WT plants treated with different topoisomerase II inhibitors. ( a ) Plants were treated with TOPII inhibitors in two ways: (i) a 2 h pulse (P) or (ii) continuous (C). In both cases, flower buds were fixed at 12 h, 28 h, or 38 h after treatment. ( b – m ) Images of pollen mother cells at different stages of meiosis of the WT treated with TOPII inhibitors. ( b ) Anaphase I treated with merbarone 1 μM (P) fixed at 38 h showing an anaphase bridge. ( c ) Anaphase I treated with merbarone 1 μM (C) fixed at 38 h showing a chromosome fragment. ( d ) Anaphase II treated with merbarone 10 μM (P) fixed at 38 h showing chromosome mis-segregation. ( e ) Telophase II treated with merbarone 1 μM (C) fixed at 38 h showing micronuclei. ( f ) Anaphase I treated with etoposide 0.05 μM (P) fixed at 38 h showing a broken anaphase bridge. ( g ) Anaphase II treated with etoposide 0.05 μM (C) fixed at 38 h showing chromosome mis-segregation. ( h ) Telophase II treated with etoposide 0.05 μM (P) fixed at 28 h showing a micronucleus. ( i ) Telophase II treated with etoposide 5 μM (P) fixed at 28 h showing a micronucleus. ( j ) Anaphase I treated with ICRF-187 0.1 μg/mL (P) fixed at 38 h showing an anaphase bridge. ( k ) Anaphase I treated with ICRF-187 100 μg/mL (C) fixed at 28 h showing two anaphase bridges. ( l ) Metaphase II/anaphase II treated with ICRF-187 100 μg/mL (C) fixed at 28 h showing an anaphase bridge. ( m ) Telophase II treated with ICRF-187 0.1 μg/mL (P) fixed at 38 h showing a micronucleus. Arrows indicate errors in meiotic divisions. Scale bar 10 μm.

Article Snippet: Etoposide (Tocris Bioscience, Bristol, UK) at 5 μM was also used in conjunction with the DSB repair assessment.

Techniques:

Journal: PLOS Biology

Article Title: Interferon regulatory factor 4 mediates nonenzymatic IRE1 dependency in multiple myeloma cells

doi: 10.1371/journal.pbio.3003096

Figure Lengend Snippet: (A) Effect of IRF4, IRE1, or XBP1 silencing on in vitro spheroid growth of AMO1. Cells were stably transfected with plasmids encoding Dox-inducible shRNAs against either IRF4 (purple) or non-targeting control (blue). Growth of these cells in the absence (closed symbols) or presence (open symbols) of Dox (0.2 μg/mL) was compared to that of cells expressing shRNAs against IRE1 or XBP1. Spheroid growth, depicted as FC confluence, was monitored by time-lapse microscopy in an IncuCyte instrument and values represent mean ± SEM. (B) Effect of IRF4, IRE1, or XBP1 silencing on number of cell divisions. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were stained with CFSE-type dye and incubated in the absence (filled curves) or presence (open curves) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dashed line) was used as a non-proliferative control. Representative experiment out of 3 independent replicates. (C) Effect of IRF4, IRE1, or XBP1 silencing on DNA replication. AMO1 shIRE1 Cl.1, shIRF4 Cl.1, or shXBP1 Cl.1 cells were pulsed with BrdU (10 μM) and incubated in the absence (filled bars) or presence (open bars) of Dox (0.2 μg/mL) and analyzed by flow cytometry. Etoposide (Eto, 25 μM, dotted line) was used as a non-proliferative control. Data represented as mean ±SEM. (D) Effect of IRE1 or IRF4 silencing on cell cycle progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence (filled symbols) or presence (open symbols) of Dox (0.2 μg/mL) for the indicated timepoints, EtOH-fixated and PI stained before analyzed by flow cytometry. The indicated cell cycle phases were determined according to univariate (DNA content) modeling. Representative experiment out of at least 3 independent replicates. (E) Effect of IRE1 or IRF4 silencing on the rate of G2/M progression. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were pre-incubated with 9 μM RO-3306 CDK1 inhibitor (synchronization to G2/M phase) in the absence or presence of Dox (0.2 μg/mL). Cells in G2/M phase were collected and their cell cycle progression during indicated time points post-sorting was analyzed by flow cytometry as before. The indicated cell cycle phases were determined according to DNA content and EdU incorporation to accurately decipher S phase. (F) Effect of IRE1 or IRF4 silencing on CDK2 activation. AMO1 shIRE1 Cl.1 or shIRF4 Cl.1 cells were incubated in the absence or presence of Dox (0.2 μg/mL) for 24 h. CDK2 was purified by immunoprecipitation. The top band is inactive CDK2 and the bottom band is the active form . Additionally, binding of the CDK2 substrate, Rb, is reduced by IRE1 or IRF4 silencing while binding of p21, the CDK inhibitor, is increased. Ig represents an isotype control for Ig detection. (G) Effect of IRE1 or IRF4 silencing on subcellular abundance of CDK2. Samples from and samples from AMO1 shIRF4 Cl.1 cells were analyzed by IB for CDK2 protein. Subcellular fractions: C—cytoplasmic, M—Membrane, SN—Soluble Nuclear, CN—Chromatin-bound Nuclear. Nuclear fractions were analyzed by IB for IRE1 and IRF4 while Cofilin, Histone H3, and Lamin B2 served as fractionation internal controls. The blots for IRE1, IRF4, Cofilin, and Lamin B2 from are shown here again for direct comparison. Data underlying this figure can be found in and .

Article Snippet: Etoposide (25 μM, #1226) was from R&D systems.

Techniques: In Vitro, Stable Transfection, Transfection, Control, Expressing, Time-lapse Microscopy, Staining, Incubation, Flow Cytometry, Activation Assay, Purification, Immunoprecipitation, Binding Assay, Membrane, Fractionation, Comparison

Journal: MedComm – Oncology

Article Title: Gene LY96 is an M2 macrophage‐related biomarker and is associated with immunosuppression in renal cell carcinoma

doi: 10.1002/mog2.52

Figure Lengend Snippet: FIGURE 6 Immunotherapy efficacy prediction and drug prediction for lymphocyte antigen 96 (LY96). (A) LY96 is positively associated with immune scores. (B–D) Comparison of LY96 expression level between ICI responder and nonresponder. (E) Twenty‐seven drugs are predicted by TAiC database for targeting LY96. (F) Seven drugs are predicted by Cellminer database. (G) Vemurafenib and etoposide are the intersections of two predictions. (H, I) Molecular docking of LY96 and two drugs. False discovery rate (FDR) or p value < 0.05 is defined as significant. ****p < 0.0001, ***p < 0.001, **p < 0.01, *p < 0.05.

Article Snippet: Etoposide (Selleck) at different concentrations (0, 1, 2, 3, 4, 5 μM) was added to the culture medium and cocultured with M2 macrophages for 24 h. Afterward, the cells were collected for further analysis.

Techniques: Comparison, Expressing

Journal: eLife

Article Title: SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

doi: 10.7554/eLife.21491

Figure Lengend Snippet: ( A ) Clonogenic assay of control SPRTN, and SPRTN -KO MEFs. Control SPRTN MEFs were treated with 4-OHT for 24 hr to induce CRE-mediated excision of SPRTN. Cells were then treated with formaldehyde, Etoposide or CPT at the indicated drug concentrations for 24 hr (72 hr for Etoposide) and subsequently allowed to form colonies for 10 days. Cells were plated in triplicate. Error bars represent S.E.M. ( B ) In vivo complex of Enzyme (ICE) assay. Schematic illustration of the method used to isolate Etoposide-induced covalent Top2 DNA-protein crosslinks from cells. ( C ) ICE assay of control SPRTN MEFs, SPRTN-KO MEFs, and conditional SPRTN MEFs reconstituted with SPRTN, SPRTN-E112A, SPRTN-Y117C, or SPRTN-∆C. Cells were either untreated or treated for 30 min with 25 uM Etoposide and immediately processed for ICE assay or processed 1 and 2 hr after washing out Etoposide (recovery). 1 ug of DNA was loaded for each time-point per cell line. Membrane was probed with an antibody against Top2 and subsequently re-probed with anti-thymine dimer antibody after 1 min UV treatment to control for DNA loading. Representative Western blot indicating SPRTN levels after reconstitution of SPRTN-KO MEFs. DOI: http://dx.doi.org/10.7554/eLife.21491.005

Article Snippet: Etoposide (Santa Cruz; CAS 33419-42-0) was dissolved in DMSO to a 50 mM stock concentration.

Techniques: Clonogenic Assay, Control, In Vivo, Membrane, Western Blot

Journal: eLife

Article Title: SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

doi: 10.7554/eLife.21491

Figure Lengend Snippet: ( A ) Chromatin fractionation of 293T-HEK cells expressing GFP-SPRTN, GFP-SPRTN-E112A, GFP-SPRTN-∆C, or GFP-SPRTN-∆C+NLS in the absence or presence of Etoposide. Proteins were separated by SDS-PAGE and transferred to a membrane for Western blot analysis with an antibody against GFP, vinculin or histone H3. DOI: http://dx.doi.org/10.7554/eLife.21491.009

Article Snippet: Etoposide (Santa Cruz; CAS 33419-42-0) was dissolved in DMSO to a 50 mM stock concentration.

Techniques: Fractionation, Expressing, SDS Page, Membrane, Western Blot

Journal: eLife

Article Title: SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

doi: 10.7554/eLife.21491

Figure Lengend Snippet: ( A ) In vitro SPRTN self-cleavage reactions. Purified proteins were incubated with or without DNA for 5 hr at 37°C. 1,10 Phe = 1,10 Phenanthroline, a zinc metal chelator and inhibitor of zinc metalloproteases. Proteins were separated by SDS-PAGE and stained with coommasie blue. ( B ) In vitro histone H3 cleavage. SPRTN was incubated with or without histone H3 (SPRTN:H3 molar ratio of 4:1) in the presence of dsDNA for the indicated time points. SPRTN-E112A or SPRTN-Y117C mutants were incubated for 2 hr. Proteins were separated on an SDS-PAGE gel and transferred to a membrane for Western blot analysis. Histone H3 cleavage as well as SPRTN self-cleavage were monitored by immunobloting with antibodies against histone H3 and SPRTN. ( C ) In vitro Top2 cleavage. Purified recombinant Top2 was pre-incubated with DNA and Etoposide to irreversibly bind Top2 to DNA. Recombinant SPRTN or SPRTN-E112A was then added alone or in combination with 10-fold molar excess of either ubiquitin or SUMO and incubated for 2 hr at 37°C. Proteins were separated by SDS-PAGE and transferred to a membrane for Western blot analysis. Membrane was stained with amido black to detect SPRTN cleavage fragments. DOI: http://dx.doi.org/10.7554/eLife.21491.008

Article Snippet: Etoposide (Santa Cruz; CAS 33419-42-0) was dissolved in DMSO to a 50 mM stock concentration.

Techniques: In Vitro, Purification, Incubation, SDS Page, Staining, Membrane, Western Blot, Recombinant, Ubiquitin Proteomics

Journal: eLife

Article Title: SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

doi: 10.7554/eLife.21491

Figure Lengend Snippet: ( A ) Comparison of SPRTN-Y117C and SPRTN-∆C mutant proteins found in patients with Ruijs-Aalfs syndrome. The NLS sequence highlights the amino acid conservation in vertebrate SPRTN species. ( B ) Fluorescent expression of GFP-SPRTN and mutant proteins in live U2OS cells. ( C ) ICE assay of DNA isolated from SPRTN control, SPRTN-KO cells or SPRTN-KO cells reconstituted with SPRTN-WT, SPRTN-∆C+NLS , or SPRTN-R408A L411A . Cells were either untreated or treated for 30 min with 25 uM Etoposide and immediately subjected to ICE assay or 1 and 2 hr after washing out Etoposide (recovery), as in . Western blot indicates SPRTN levels after reconstitution of SPRTN-KO MEFS. ( D ) Representative flow cytometry analysis of control LCL cells and patient (B-II-1) LCL cells treated with formaldehyde, Etoposide or CPT for 0, 1 or 4 hr. Cells were collected at indicated time points and processed for γ-H2AX staining. Untreated controls are indicated in black for comparison to treated cells. 10,000 cells were scored for each experiment. ( E ) ICE assay of DNA isolated from control or B-II-1 cells that were either untreated or treated with 25 uM Etoposide for the indicated times as in and . DOI: http://dx.doi.org/10.7554/eLife.21491.011

Article Snippet: Etoposide (Santa Cruz; CAS 33419-42-0) was dissolved in DMSO to a 50 mM stock concentration.

Techniques: Comparison, Mutagenesis, Sequencing, Expressing, Isolation, Control, Western Blot, Flow Cytometry, Staining

Journal: eLife

Article Title: SPRTN is a mammalian DNA-binding metalloprotease that resolves DNA-protein crosslinks

doi: 10.7554/eLife.21491

Figure Lengend Snippet: DNA-protein crosslinks may arise stochastically as a consequence of normal metabolic processes occurring at or in the vicinity of DNA or by exogenous sources such as Etoposide. DPCs are resolved by SPRTN to allow for unperturbed and timely DNA replication. When SPRTN is absent or when its activity is compromised, such as in patients with Ruijs-Aalfs syndrome (SPRTN-Y117C or SPRTN-∆C), DPCs can persist and perturb DNA replication and transcription (not shown). DPCs, if not resolved, can lead to genomic instability, tumorigenesis and contribute to accelerated aging. DOI: http://dx.doi.org/10.7554/eLife.21491.013

Article Snippet: Etoposide (Santa Cruz; CAS 33419-42-0) was dissolved in DMSO to a 50 mM stock concentration.

Techniques: Activity Assay

Journal: Biology of reproduction

Article Title: Development of a short-term fluorescence-based assay to assess the toxicity of anticancer drugs on rat stem/progenitor spermatogonia in vitro.

doi: 10.1095/biolreprod.110.083568

Figure Lengend Snippet: FIG. 6. Effects of chemotherapeutic agents, cisplatin, etoposide, and bleomycin on in vitro cultured rat EGFPþ clusters. For cytotoxicity assays, clusters were first grown for 4 days without drug. At Day 4, culture medium was changed and replaced by a fresh one with drug or vehicle. Following culture for 2 days, rat EGFPþ clusters were fixed and then counted using an automated fluorescence microscope, as described in Materials and Methods. The cytotoxicity of each drug for rat EGFPþ clusters was assessed by plotting concentration-response curves for the cluster numbers (left axis) and surface area (right axis). Clusters in 96-well plates were treated with cisplatin (A), etoposide (B), bleomycin (C), or a combination of the three drugs (D). Cluster number and surface area were determined 48 h later. Data shown represent the average of three independent experiments, each containing three replicates. The data are presented as the means 6 SEM.

Article Snippet: Cisplatin, etoposide, and bleomycin were purchased from LKT Laboratories.

Techniques: In Vitro, Cell Culture, Fluorescence, Microscopy, Concentration Assay

Journal: bioRxiv

Article Title: Break-induced replication drives large-scale genomic amplifications in cancer cells

doi: 10.1101/2024.08.27.609980

Figure Lengend Snippet: a , FACS profiles showing de novo DNA synthesis (BrdU incorporation) in U2OS cells with greater than G2/M DNA content (as assessed by 7AAD staining) 48 or 72 h following exposure to 9 Gy. Cells were pulsed with BrdU for 1 h prior to harvest. b , Representative FACS profiles (PI staining) of U2OS cells 48 h following exposure to 9 Gy with or without nocodazole (Noc) treatment at the 24-48 h time point following IR exposure. Cells with genomic amplifications highlighted in red. Quantitation is shown in . c , Elisa-based quantitation of BrdU incorporation in the peak fractions of DNA (50 ng each of L:L, H:L, and H:H DNA) isolated from the CsCl ultracentrifugation gradient shown in . d , Workflow ( top ) and representative FACS profiles (PI staining; bottom ) of U2OS cells left untreated or exposed to IR for 48 h and treated with or without aphidicolin (Aph) added immediately following IR and washed 24 h after (0-24 h) or added 24 h post-IR (24-48 h). Cells with genomic amplifications are highlighted in red, and quantitation of the results is shown in . e , The appearance of H:H DNA in cells exposed to IR is aphidicolin-sensitive. The line histogram is an extension of the plot shown in , and shows additional treatment with aphidicolin (Aph) with or without IR treatment as depicted in the experimental workflow shown on top. f , Histogram showing the percentage of U2OS cells with genomic amplifications (as determined by PI-FACS) 72 h following treatment with etoposide (1 μg ml - ), doxorubicin (0.1 μM), or exposure to ultraviolet radiation (UV; 100 J m - ). Data represent the average of three independent experiments ± S.D. g , Work flow ( top ) and quantitation of the percentage of A si SI-ER-U2OS cells with genomic amplifications (as determined by PI-FACS) following the induction of DSBs by the addition of 4-OHT for 48 h. Cells were treated with or without aphidicolin (Aph) added together with 4-OHT and washed 24 h after (0-24 h), or with Aph added 24 or 48 h following treatment with 4-OHT and harvested 24 h after Aph (48 and 72 h following treatment with 4-OHT, respectively). Data represent the average of three independent experiments ± S.D. ** p < 0.01. h , Representative FACS profiles showing de novo DNA synthesis (BrdU incorporation) in A si SI-ER-U2OS cells with greater than G2/M DNA content (as assessed by 7AAD staining) 48, 72, or 96 h following treatment with 300 nM 4-hydroxy-tamoxifen (4-OHT). Cells were pulsed with BrdU for 1 h prior to harvest.

Article Snippet: The DNA LIG4 inhibitor SCR7 (#SML1546, Sigma Aldrich) was added 24 h before irradiation, and was replenished every 24 h. Analysis of rereplication induction by FACS (PI staining) following the induction of DNA damage by etoposide (1 μg ml - , TOCRIS), doxorubicin (0.1 μM, Sigma Aldrich), or by UV (100 J m - ) was performed 72 following treatment.

Techniques: DNA Synthesis, BrdU Incorporation Assay, Staining, Quantitation Assay, Enzyme-linked Immunosorbent Assay, Isolation