Journal: Cancer science

Article Title: Steroid-Modulated Transcription Synergistically Forms DNA Double-Strand Breaks With Topoisomerase II Inhibitor.

doi: 10.1111/cas.70081

Figure Lengend Snippet: FIGURE 3 | MPA promotes DSB formation only when combined with Top2 inhibitors that form a Top2-covalent complex. (A) siRNA-mediated knockdown efficiency of Top2α in HeLa cells was analyzed by western blotting. (B) Quantities of DSBs in Top2α-silenced and 3 μM MPA-treated HeLa cells combined with 0.3 μM ETP for 24 h were analyzed by evaluating the γH2AX intensity using a flow cytometer. The representative distribu- tions are shown in the left panel. (C) MPA-induced DSB formation (γH2AX) when combined with ETP was calculated as in the left panel at different Top2 expression levels (siCtrl or siTop2A) in (B). (D) Quantities of nuclear-localized Top2α in 3 μM MPA-treated HeLa cells combined with 0.3 μM ETP for 8 h were analyzed. Representative distributions are shown. (E) Quantities of DSBs in3 μM MPA-treated HeLa cells combined with various Top2 inhibitors, including 0.3 μM ETP, 0.03 μM DOX, 20 μM merbarone (MER), 1 μM ICRF-193 (ICRF), and 10 μM novobiocin (NOVO) for 24 h were analyzed by evaluating the γH2AX intensity using a flow cytometer. Data represent means ± SD of at least three independent experiments.

Article Snippet: Thereafter, the samples were washed with PBS, blocked in 3% BSA for 1 h, and incubated with PE- conjugated Top2α antibody (1:100, 34,184, CST) for 1 h at room temperature.

Techniques: Knockdown, Western Blot, Flow Cytometry, Expressing

Journal: Cancer science

Article Title: Steroid-Modulated Transcription Synergistically Forms DNA Double-Strand Breaks With Topoisomerase II Inhibitor.

doi: 10.1111/cas.70081

Figure Lengend Snippet: FIGURE 4 | MPA promotes DSB formation in combination with ETP in the vicinity of promoter regions. (A) Location of DSB formation in 10 μM MPA-treated HeLa cells combined with 1 μM ETP was detected by BLISS analysis. The feature distribution was shown using ChIPseeker. (B) Quantities of DSBs in 1 μM ETP-treated HeLa cells under 100 μM DRB treatment for 8 h were analyzed by evaluating the γH2AX intensity using a flow cytometer. (C and D) 10 μM MPA-induced DSBs (C) and nuclear-localized Top2α (D) in HeLa cells when combined with 1 μM ETP under 100 μM DRB treatment for 8 h were analyzed using a flow cytometer. (E) RNA synthesis in HeLa cells treated as indicated was analyzed by EU incorporation using a flow cytometer. Cells were treated with 100 μM DRB and 3 μM MPA 1 h before adding EU, and treatment was continued until the assay. Data represent means ± SD of at least three independent experiments. (F) Volcano plot of MPA-modulated gene expression is shown. The genes with |fold change| ≥ 1.3 and p ≤ 0.01 were considered differentially expressed. The pie chart shows the number of up-regulated and down-regulated DEGs. The arrow indicates the most variable gene (GMPR) in the up-DEGs. (G) Peak profiles around the GMPR locus for ETP and ETP + MPA treatment in the BLISS data are shown. #1 and #2 are the results of two independent experiments.

Article Snippet: Thereafter, the samples were washed with PBS, blocked in 3% BSA for 1 h, and incubated with PE- conjugated Top2α antibody (1:100, 34,184, CST) for 1 h at room temperature.

Techniques: Flow Cytometry, Gene Expression

Journal: Cancer science

Article Title: Steroid-Modulated Transcription Synergistically Forms DNA Double-Strand Breaks With Topoisomerase II Inhibitor.

doi: 10.1111/cas.70081

Figure Lengend Snippet: FIGURE 5 | Steroids increase DSBs when combined with ETP. (A) Quantities of DSBs in steroid-treated HeLa cells combined with 0.3 μM ETP for 24 h were analyzed by evaluating γH2AX intensity using a flow cytometer. All 54 steroids were used at a concentration of 3 μM. (B) Steroid-induced DSBs when combined with ETP were calculated in (A). (C) Schematic model of steroid-induced synergistic effect with ETP is shown. Top2 molecules trapped in DNA by etoposide are partially released under etoposide monotreatment (left part). In contrast, when steroids are combined with etopo- side, steroid-modulated transcriptional activity collides with the trapped Top2 molecules, which are converted to Top2ccs, resulting in DSB forma- tion and cytotoxicity (right part). This figure was created with BioRender.com.

Article Snippet: Thereafter, the samples were washed with PBS, blocked in 3% BSA for 1 h, and incubated with PE- conjugated Top2α antibody (1:100, 34,184, CST) for 1 h at room temperature.

Techniques: Flow Cytometry, Concentration Assay, Activity Assay

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: (A) The mRNA levels of TOP2A in normal tissues (n=13) and CC tissues (n=306) were compared using the GEPIA database. (B) RT-qPCR analysis was performed to validate the expression of Top2α in 21 normal tissues (Normal) and 20 cervical cancers (Cancer). The relative mRNA levels were calculated using the 2 -ΔΔCT method. EEF1A1 was used as the internal control for data normalization. (C) Representative immunohistochemical staining of Top2α in 10 normal tissues, 10 cervical intraepithelial neoplasia, and 10 cervical cancers. (D) Western blot analysis of Top2α levels were characterized in primary human foreskin keratinocytes (HFKs), HFK16 (HPV16 positive), HFK31 (HPV31 positive), and CIN612 (HPV31 positive) (left). Top2α protein levels were tested by western blot in HaCaT, HaCaT16 (HPV16 positive), and HaCaT31 (HPV31 positive) cells (right). GAPDH served as the loading control. Similar results were seen in three independent experiments. The statistical analysis was assayed by 2-tailed t test. * P ≤0.05, **** P ≤0.0001. (E) Western blot analysis was performed using antibodies of Top2α and GAPDH as the loading control. (F-H) RT-qPCR was performed to detect TOP2A, E6, and E7 mRNA levels in E6, E7, and E6/E7-overexpressing cells.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: Quantitative RT-PCR, Expressing, Control, Immunohistochemical staining, Staining, Western Blot

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: (A) The Top2α knockdown efficiency was confirmed by western blot analysis in the CIN612 cell background. GAPDH served as the loading control. (B) DNA lysates in Top2α knockdown cells was harvested at 0 hours and 72 hours. Southern blot analysis was performed to examine viral episomes using the HPV31 genome as a probe. Integrated, nicked/circular, linearized, and supercoiled/episomal genomes are indicated. (C-E) HPV31-positive HaCaT31 cells were transduced with lentivirus containing control scramble shRNA sequences (TRC) or two shRNA sequences for Top2α (shTop2α#1 and shTop2α#2). The protein and DNA were extracted from HaCaT31-TRC, shTop2α#1, and shTop2α#2 cell lines. (C) Western blot analysis was carried out to compare Top2α, expression in HaCaT31-TRC, shTop2α#1, and shTop2α#2 cell lines, with GAPDH as the loading control. (D) DNA-qPCR was performed to measure the HPV31 E6 levels. Mitochondrial DNA (mtDNA) was used as the internal control for data normalization. The data were representative from three independent experiments. The statistical analysis was assayed by 2-tailed t test. Data are means ± standard errors. ** P ≤0.01. (E). Representative dot blot analysis of undifferentiated HPV31-containing keratinocytes with Top2α depletion.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: Knockdown, Western Blot, Control, Southern Blot, Transduction, shRNA, Expressing, Dot Blot

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: (A) The comet assay in HaCaT31 cell lines that were treated with DMSO, PluriSln#2 (Top2α inhibitor, 10 μM), or etoposide (TOP2 inhibitor, 50 μM) for 6 hours. Quantitation of percent tail versus nucleoid body is shown in the violin plots (n=at least 50 number of nucleoids, ns=no significance, **** P ≤0.0001, Scale bar, 100 μm). (B) Comet assays for DNA break formation in HaCaT31 cells with TRC, shTop2α#1, and shTop2α#2. Quantitation of tail percentage versus nucleoid body is shown in the violin plots (n=at least 50 number of nucleoids, ns=no significance, Scale bar, 100 μm). (C) Western blot analysis was performed using antibodies to γH2AX, ATM, phosphorylated ATM, ATR, phosphorylated ATR, DNA-PKcs, and phosphorylated DNA-PKcs in Top2α-deficient cells. GAPDH served as the loading control. Data were representative of at least three independent biological repeats.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: Single Cell Gel Electrophoresis, Quantitation Assay, Western Blot, Control

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: (A) RNA-seq analysis was performed using HaCaT31 cells with Top2α knockdown. 117 differential expressed genes were identified as downstream targets of Top2α. Top20 pathways regulated by Top2α were listed with Metascape online analysis. (B) RT-qPCR analysis was conducted to assess the expression levels of TOP2A, IL-1B, IL-6, TNF-α, IL-8, IL-18, IL-17, CCL2, CCL5, CCL20, CXCL9, CXCL10, CXCL11, and IFNB in HaCaT31 cells with Top2α knockdown. GAPDH was used as the internal control for data normalization. Data are representative of at least three independent biological repeats. The statistical analysis was assayed by 2-tailed t test. * P ≤0.05, ** P ≤0.01, *** P ≤0.001.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: RNA Sequencing, Knockdown, Quantitative RT-PCR, Expressing, Control

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: (A) The protein microarray was utilized to measure the levels of IL-6, IL-8, and CCL2 in vaginal discharge samples from normal, cervical intraepithelial neoplasia (CIN), and cervical cancer patients. (B) Gene Expression Profiling Interactive Analysis (GEPIA) was performed to analyze the correlation of IL-6, IL-8, and CCL2 expression with the overall survival rate of cervical cancer patients. The red lines represent patients with high gene expression, and the blue lines with low gene expression. (C) Western blot demonstrating the expression of Top2α, IL-6, IL-8, and CCL2 proteins in the Top2α-depleted cell lines. GAPDH served as the loading control. (D) The levels of secreted IL-6, IL-8, and CCL2 outside Top2α knockdown cells were determined by ELISA assay. Data are representative of at least three independent biological repeats. The statistical analysis was assayed by 2-tailed t test. * P ≤0.05, ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: Microarray, Gene Expression, Expressing, Western Blot, Control, Knockdown, Enzyme-linked Immunosorbent Assay

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: (A) Heat maps of inflammatory regulatory genes regulated by Top2α knockdown. TRC_rep1 and TRC_rep_2 are replicates of scramble HaCaT31 cells, while shTop2α#1 (rep1 and rep2) and shTop2α#2 (rep1 and rep2) are replicates of HaCaT31 cells with Top2α knockdown respectively. (B) RT-qPCR was performed to detect TLR4 mRNA levels in Top2α knockdown cells. (C) Western blot analysis was performed using antibodies of Top2α, TLR4, and NF-κB, with GAPDH as the loading control. (D) The TLR4 promoter plasmid were transfected into HaCaT31-TRC, shTop2α#1, and shTop2α#2 cell lines. A pRL-TK plasmid was transfected to normalize the transfection efficiency data. Luciferase activity was measured in cell lysates for 48 hours post-transfection. Data are exhibited as means ± SEM, and are representative of at least two biological independent repeats. ** P ≤0.01. (E and F) ChIP-qPCR was performed in Top2α-depleted cells to identify the binding of Top2α on the TLR4 promoter regions. IgG served as an antibody control. (G) IL-6 and IL-8 were quantified in the supernatants from outside of HaCaT31 cells with TAK-242 treatment of indicated concentrations. ** P ≤0.01, *** P ≤0.001, **** P ≤0.0001. (H and I) The viral DNAs extracted from HaCaT31 cells treated with DMSO or TAK-242 (10 nM) for 72 hours were visualized by dot blot or DNA-qPCR. Data are representative of three biological independent repeats. ns=no significance, *** P ≤0.001.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: Knockdown, Quantitative RT-PCR, Western Blot, Control, Plasmid Preparation, Transfection, Luciferase, Activity Assay, ChIP-qPCR, Binding Assay, Dot Blot

Journal: bioRxiv

Article Title: Topoisomerase IIα orchestrates secretion of IL-6 and IL-8 with human papillomavirus replication

doi: 10.1101/2025.04.03.647014

Figure Lengend Snippet: HPV induces Top2α, partially through E6, which then regulates TLR4 transcription by directly binding to TLR4 promoter region. The elevated TLR4 signaling facilitates the secretion of IL-6/IL-8 that in turn promotes HPV replication. Inhibition of Top2α by shRNA approaches, suppression of TLR4 by TAK-242, and neutralization of IL-6/IL-8 significantly diminishes HPV replication. The image is drawn using Biorender software.

Article Snippet: TLR4 promoter reporter plasmid was transfected into Top2α-silencing cells using Attractene Transfection Reagent (Qiagen, #301005) for 48 hours.

Techniques: Binding Assay, Inhibition, shRNA, Neutralization, Software

Journal: Biomedicines

Article Title: CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci

doi: 10.3390/biomedicines12071514

Figure Lengend Snippet: Top2α mediates resistance to CX-5461 in mice. ( A ) Schematic of Top2α mutations. The mouse Top2α gene is shown with the N-terminal ATPase, catalytic TOPRIM, DNA-binding winged-helix domain (WHD), and C-terminal domain (CTD) domains highlighted. Seven different Top2α mutations were identified by whole exome sequencing (WES) of DNA extracted from tumors that had acquired resistance to CX-5461 in vivo. Frameshift, missense and nonsense mutations are denoted by stars, circles, and hexagons, respectively. This figure is adapted from Wendorff et al. . ( B ) Top2α mRNA levels in T2A WT and T2A +/K1266 cells. Basal Top2α mRNA abundance in the T2A WT and T2A +/K1266 cells was measured by real-time quantitative PCR (qPCR) and normalized to B2M expression. Error bars represent standard deviation (* p < 0.05, n = 3). ( C ) Top2α protein expression in T2A WT and T2A +/K1266 cells. Representative Western blot of basal Top2α protein. ( D ) The representative image of decatenation assays demonstrating that T2A WT cells have greater cellular Top2 activity than T2A +/K1266 cells. The nuclear extracts containing Top2 enzymes are isolated from each group of cells and are serially diluted before incubation with kDNA. In both instances shown, the Top2 activity as measured by the decatenated fraction of each lane is greater in the lower dilutions of the T2A WT cells than the T2A +/K1266 cells. ( E ) CX-5461 in vitro dose–response assay. T2A WT (green line) and T2A +/K1266 (red line) cells were treated with 0.3 nM–1 μM CX-5461 for 24 h in triplicate, and the live cell number was counted by volumetric FACS of propidium iodide (PI)-negative cells. Results were normalized to the number of live cells after drug vehicle treatment (100%), and the line of best fit was plotted. Concentrations of the drug inhibiting 50% of growth (GI 50 ) were calculated using GraphPad Prism 10. ( F , G ) The Kaplan–Meier curve of mice transplanted with T2A WT ( F ) and T2A +/K1266 ( G ) cells ± CX-5461. Mice were treated every three days with 35 mg/kg of CX-5461. Median survival: T2A WT = 15 days, T2A +/K1266 = 1 day (*** p < 0.001, n.s. = not significant, n = 8/group).

Article Snippet: When the Top2 inhibitory activity of drugs was tested, recombinant Top2α enzyme (TopoGen, Buena Vista, CO, USA) was used in place of cell-extracted Top2.

Techniques: Binding Assay, Sequencing, In Vivo, Real-time Polymerase Chain Reaction, Expressing, Standard Deviation, Western Blot, Activity Assay, Isolation, Incubation, In Vitro

Journal: Biomedicines

Article Title: CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci

doi: 10.3390/biomedicines12071514

Figure Lengend Snippet: Top2α determines the level of activation of DDR pathways in CX-5461-treated cells but not the ability of CX-5461 to inhibit Pol I transcription. ( A ) T2A WT and T2A +/K1266 cells were incubated with radiolabeled orthophosphate for 20 min and then treated with varying concentrations of CX-5461 as indicated. RNA was isolated and analyzed on agarose-formaldehyde gel. Newly synthesized 47 pre-rRNA was detected by autoradiography (a representative experiment is shown; upper panel) and total 28S rRNA was detected by ethidium bromide staining (a representative experiment is shown; bottom panel). ( B ) Representative Western blots showing the downstream signaling response to CX-5461 ± KU-55933 treatment of T2A WT and T2A +/K1266 cells. Cells were treated with 30 nM CX-5461 for 30–180 min ± 30 min pre-treatment with ATM inhibitor KU-55933. Western blots were probed for phosphorylated ATM (S1981), phosphorylated p53 (S15), total p53 and loading controls tubulin and β-actin. ( C , D ) Quantitative real-time PCR of ( C ) p21 and ( D ) Puma from the experiment outlined in ( B ). Error bars represent standard deviation (* p < 0.05, ** p < 0.01, *** p < 0.001, n = 5). Dotted line represents average relative expression value at time point 0. ( E ) T2A WT and T2A +/K1266 cells were treated with varying concentrations of CX-5461 (as in E) for 24 h after pre-treatment with ±1 μM KU-55933 for 30 min in triplicate. Cell viability was determined by measuring absorption at 570 nm after incubation with Alamar Blue. Results were normalized to vehicle-treated cells (set 100%), and GI 50 values were determined and plotted as a bar graph. Error bars represent standard deviation (n = 3).

Article Snippet: When the Top2 inhibitory activity of drugs was tested, recombinant Top2α enzyme (TopoGen, Buena Vista, CO, USA) was used in place of cell-extracted Top2.

Techniques: Activation Assay, Incubation, Isolation, Synthesized, Autoradiography, Staining, Western Blot, Real-time Polymerase Chain Reaction, Standard Deviation, Expressing

Journal: Biomedicines

Article Title: CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci

doi: 10.3390/biomedicines12071514

Figure Lengend Snippet: CX-5461 does not induce significant formation of Top2α (Top2α-cc) cleavage complexes, but causes low-level, nucleolar-localized DNA damage. ( A ) Schematic of the TARDIS assay. Cells were suspended in a thin layer of agarose on a microscope slide. After lysis with SDS and incubation with a high salt buffer, only DNA and macromolecules covalently bound to the DNA remain on the slide. Covalently bound proteins, such as active Top2α can be imaged by immunofluorescent labelling. ( B ) Representative images of Eμ- Myc T2A WT cells pre-treated with 1 μM MG-132 for 15 min to prevent proteolytic degradation of DNA-bound Top2α, then treated with drug vehicle (top panel), 30-nM CX-5461 (middle panel), or 50-nM doxorubicin (bottom panel) for 2 h. The DNA is stained blue by DAPI, and Top2α bound covalently to DNA is shown in green. Scale bar, 5 μm ( C ) Quantitation of the experiment is outlined in ( B ). While CX-5461 exhibits a similar degree of Top2 inhibition via the decatenation assay , CX-5461 causes significantly fewer Top2α-DNA complexes in cells than doxorubicin (n = 45–47 cells, *** p < 0.001). ( D ) Representative images of Eμ- Myc T2A WT cells treated with either drug vehicle, 30 nM CX-5461, or 60nM Etoposide for 1 h immunostained for the nuclei (DAPI; blue), the nucleoli (Fibrillarin; green) and γH2AX (red). ). Scale bar, 10.4 μm. ( E ) Quantitation of the distance to the closest fibrillarin spot from each γH2AX foci. Whiskers extend to the 10th and 90th percentiles, and the mean is represented by the cross. CX-5461-treated cells exhibit a shorter median γH2AX-Fib distance compared to vehicle- or etoposide-treated cells (** p < 0.01). ( F ) Quantitation of the proportion of sub-0.3 μm γH2AX-Fib distances for both CX-5461 and etoposide-treated samples from three independent experiments. Error bars represent the standard deviation (* p < 0.05).

Article Snippet: When the Top2 inhibitory activity of drugs was tested, recombinant Top2α enzyme (TopoGen, Buena Vista, CO, USA) was used in place of cell-extracted Top2.

Techniques: Microscopy, Lysis, Incubation, Staining, Quantitation Assay, Inhibition, Standard Deviation

Journal: Biomedicines

Article Title: CX-5461 Preferentially Induces Top2α-Dependent DNA Breaks at Ribosomal DNA Loci

doi: 10.3390/biomedicines12071514

Figure Lengend Snippet: CX-5461 treatment induces targeted, Top2α-mediated DNA damage at the rDNA loci. ( A ) HTETOP cells were grown for 24 h either untreated (−Dox) or in the presence of 1 mg/mL Doxycycline (+Dox) and then treated with ±2 μM CX-5461 for 1 h before the DNA double-strand breaks (DSB) were labelled with BrdUTP using terminal transferase. DNA was extracted and sonicated to 400–600 bps fragments, and the labelled DNA was enriched by anti-BrdUTP antibodies. DNA enrichment was quantitated by qPCR. Signal was normalized to control (IgG) and plotted as fold changes. Error bars represent the standard deviation (n = 3). ( B ) HTETOP cells were grown for 24 h either untreated (−Dox) or in the presence of 1 mg/mL Doxycycline (+Dox) and then treated with ±5 µM etoposide for 2 h. Samples were processed and analyzed as in ( A ). Error bars represent the standard deviation (n = 3). ( C ) HTETOP cells were grown for 24 h either untreated (-Dox) or in the presence of 1 mg/mL Doxycycline (+Dox) and then treated with ±3 nM Actinomyci D (ActD) for 4 h. Samples were processed and analyzed as in ( A ). Error bars represent the standard deviation (n = 3). ( D ) HTETOP cells were grown for 24 h untreated (no Dox) and then treated with 2 μM CX-5461 for different period of time as indicated. Samples were processed and analyzed as in ( A ). Error bars represent the standard deviation (n = 3).

Article Snippet: When the Top2 inhibitory activity of drugs was tested, recombinant Top2α enzyme (TopoGen, Buena Vista, CO, USA) was used in place of cell-extracted Top2.

Techniques: Sonication, Control, Standard Deviation

Journal: Cell death & disease

Article Title: MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53-independent manner.

doi: 10.1038/s41419-024-06474-3

Figure Lengend Snippet: Fig. 9 Working model. A working model for MDM2 provides TOP2 poison resistance by promoting proteolysis of TOP2βcc in a p53- independent manner.

Article Snippet: The following antibodies (Abs) were used: MDM2 (OP46, Calbiochem, 1:1000), MDM2 (86934 S, Cell Signaling Technology, 1:500), TOP2β (611493, BD Biosciences, 1:1000), TOP2β (sc-25330, Santa Cruz, 1:1000), TOP2α (12286, Cell Signaling Technology, 1:1000), p53 (2527 S, Cell Signaling Technology, 1:1000), p21 (2947 S, Cell Signaling Technology, 1:1000), p-CHK1 (2348, Cell Signaling Technology, 1:2000), CHK1 (sc-8408, Santa Cruz, 1:1000), ACTIN (A5441, Sigma, 1:10000), β-TrCP1 (4394, Cell Signaling Technology, 1:1000), FLAG (F1804, Sigma, 1:2000), γH2AX (05-636, Millipore, 1:1000), PARP (9542, Cell Signaling Technology, 1:1000), Caspase 3 (9665, Cell Signaling Technology, 1:1000), HA (11867423001; Roche; 1:2000).

Techniques: