Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 1. TOP2A co-expression patterns and associated biological pathways. (A) Non-negative matrix decomposition (NMF) clustering was performed and the best consensus clustering values for the two subgroups were determined. (B) Analysis of overall survival (OS) of 490 LUAD patients in the TCGA cohort based on two TOP2A co-expression clusters. (C,D) Heat map showing representative Hallmark and KEGG pathways in the two TOP2A co-expression clusters. (E,F) Kaplan–Meier curves for high and low TOP2A co-expression- associated gene subgroups of patients; (E) test cohort (TCGA-LUAD); (F) GEO cohort (GSE11969); (G,H) ROC curves showing TOP2A co-expression associated gene risk scores for predictive efficiency of 1-, 3-, and 5-year survival; (G) test cohort (TCGA-LUAD); (H) GEO cohort (GSE11969).

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Expressing

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 2. Relationship between TOP2A co-expression-associated gene risk cohort and LUAD tumor mutation burden and immune microenvironment. (A) Mutation profile of SMGs in TCGA-LUAD grouped by low and high TOP2A co-expression-associated gene scores. Each column contains a proxy for individual patients. (B) Relative distribution of tumor mutation burden in subgroups with high versus low TOP2A co-expression- associated gene scores. (C) Correlation between TMB and TOP2A co-expression-associated gene risk scores and their distribution in low- and high-risk groups. (D) Kaplan–Meier curves for high and low risk groups of TMB and TOP2A co-expression-associated genes. (E) Proportion of tumor-infiltrating immune cells in the high- and low-risk groups of TOP2A co-expression-associated genes using the CIBERSORT algorithm. In each group, scattered dots represent the expression values of tumor microenvironment cells. The thick line represents the median value. The bottom and top of the box are the 25th and 75th percentiles (interquartile range). Statistical differences were compared by Wilcoxon rank sum test as follows. *p < 0.05; **p < 0.01; ***p < 0.001.

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Expressing, Mutagenesis

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 3. TOP2A co-expression-associated gene risk scores predict the efficacy of immunotherapy. (A) Association of TOP2A co-expression-associated gene risk scores with 11 immune checkpoints, with red circles representing positive associations and blue circles representing negative associations. (B) Distribution of four classical clinical immune checkpoint molecules in high and low risk of TOP2A co-expression-associated genes. (C) Proportion of patients with clinical response to anti-PD1 immunotherapy in the low or high TOP2A co-expression-associated gene score groups. CR/PR vs. SD/PD: 71% vs. 29% in the low VMRG score group; 89% vs. 11% in the high VMRG score group. (D) Functional distribution of immune dysfunction and immune escape in the high and low risk groups of TOP2A co-expression-related genes and the relative distribution of TIDE. (E) Relative distribution of TIDE in the high and low CD8+ T cell infiltration groups. (F) CD8+ T cell infiltration combined with high and low risk groups to analyze the relative distribution of TIDE. (G) CD8+ T-cell infiltration was integrated with high- and low-risk groups to analyze the proportion of patients with clinical responses to PD1 immunotherapy. CR/PR vs. SD/PD: 75% vs. 25% in the low-risk group; 86% vs. 14% in the high-risk group.

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Expressing, Functional Assay

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 4. TOP2A regulates the proliferative capacity of non-small cell lung cancer. (A,B) Stable overexpression and knockdown systems of TOP2A identified by WB and qPCR. Some blot strips were cut prior to hybridization with the antibody, and the original blot strips are listed in the supplemental material (Supplementary Original western blot images). (C,D) Proliferation capacity of A549 and H1299 cells after overexpression or knockdown of TOP2A was detected by CCK-8 assay. (E–H) Colony formation analysis of non-small cell lung cancer cell growth. Data were determined by three replicate experiments (mean ± standard deviation).

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Over Expression, Knockdown, Hybridization, Western Blot, CCK-8 Assay, Standard Deviation

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 5. TOP2A regulates apoptosis and cycle in non-small cell lung cancer. (A,B) Flow cytometry to detect apoptosis after A549 and H1299 transfection with Si-TOP2A. (C,D) Flow cytometry to detect apoptosis after A549 and H1299 transfection with OE-TOP2A. (E,F) Flow cytometry to detect the cell cycle of A549 and H1299 after transfection with Si-TOP2A.

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Flow Cytometry, Transfection

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 6. TOP2A expression was significantly correlated with PD-L1 expression and VM formation in NSCLC tissues. (A) The presence of VM was detected by CD31/PAS double staining. CD31+PAS− channels are endothelium-dependent vessels (red arrows). (B) CD31−PAS+ channels are considered VM (blue arrows), in which erythrocytes are visible (black arrows). (C) Typical NSCLC adjacent to normal and tumor tissue specimens with representative TOP2A staining (400 ×). (D) Immunohistochemical methods to detect VM expression in TOP2A-positive and TOP2A-negative groups. (E) Kaplan–Meier survival analysis showed poor overall survival in patients with high VM expression. (F) Representative PD-L1 immunohistochemical staining in lung adenocarcinoma and lung squamous carcinoma (200 ×). (G) Detection of PD-L1 expression in TOP2A- positive and TOP2A-negative groups by immunohistochemistry.

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Expressing, Double Staining, Staining, Immunohistochemical staining, Immunohistochemistry

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 7. TOP2A is involved in regulating the cytoskeleton of NSCLC and VM formation in A549 and H1299 cells in vitro. (A) The relationship between EMT and TOP2A expression was analyzed by TCGA-LUAD (n = 465). (B) The effect of TOP2A knockdown on the cytoskeleton of A549 and H1299 cells was detected by Comas Brilliant Blue staining. (C) FITC-ghost pen cyclic peptide immunofluorescence assay the effect of overexpression of TOP2A on the cytoskeleton of A549 and H1299 was detected. (D,E) Tube formation assay showed that overexpression of TOP2A promoted the formation of VM in A549 and H1299 cells. PAS staining of vascular-like structures in Matrigel showed a significant decrease in the total area and total length of tube formation in the TOP2A overexpression group. (F,G) Western blotting was performed to detect the expression of VM formation-related proteins in A549 and H1299 cells. Some blot strips were cut prior to hybridization with the antibody, and the original blot strips are listed in the supplemental material (Supplementary Original western blot images).

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: In Vitro, Expressing, Knockdown, Staining, Immunofluorescence, Over Expression, Tube Formation Assay, Western Blot, Hybridization

Journal: Scientific reports

Article Title: The role of TOP2A in immunotherapy and vasculogenic mimicry in non-small cell lung cancer and its potential mechanism.

doi: 10.1038/s41598-023-38117-6

Figure Lengend Snippet: Figure 8. Knockdown of Wnt3a counteracted the TOP2A-induced invasive phenotype. (A) Enrichment analysis of TCGA (n = 585) and GSE116959 (n = 68) revealed that the angiogenic signaling pathway was significantly enriched in the Wnt3a high expression group. (B) Immunohistochemical methods to detect VM expression in the Wnt3a positive and Wnt3a negative groups. (C,D) The results of tube formation assay showed that Wnt3a knockdown significantly inhibited the VM formation ability of A549 and H1299 cells compared with the TOP2A overexpression group. (E) Wnt3a knockdown significantly inhibited the colony formation ability of non-small cell lung cancer cells compared with the control group. (F) Western blot detection of VM in overexpressed TOP2A cells before and after knockdown of Wnt3a expression of formation-related proteins. Some blot strips were cut prior to hybridization with the antibody, and the original blot strips are listed in the supplemental material (Supplementary Original western blot images).

Article Snippet: After PVDF membranes were closed with 5% skimmed milk for 1.5 h at room temperature, PVDF membranes were incubated with diluted primary antibody overnight at 4 °C in a shaker. (TOP2A and wnt3a primary antibodies were from Abcam, all other antibodies were from Proteintech.)

Techniques: Knockdown, Expressing, Immunohistochemical staining, Tube Formation Assay, Over Expression, Control, Western Blot, Hybridization

Journal: Molecular Pharmacology

Article Title: Small Molecule Inhibitors Confirm Ubiquitin-Dependent Removal of TOP2-DNA Covalent Complexes

doi: 10.1124/mol.119.118893

Figure Lengend Snippet: Effect of inhibiting E1 ubiquitin-activating enzyme on TOP2-DNA complex levels measured using the TARDIS assay. (A and B) K562 cells were treated for 2 hours with etoposide alone or in combination with 10 µM MG132, 10 µM MLN7243, or 10 µM MG132 and 10 µM MLN7243. Cells were incubated for up to a further 2 hours in etoposide-free medium but in the continued presence of DMSO, 10 µM MG132, or 10 µM MLN7243. Levels of TOP2A- and TOP2B-DNA complexes were measured 0, 0.5, 1, and 2 hours after etoposide removal using the TARDIS assay. Median normalized integrated fluorescence values of three independent experiments are shown (circle symbols) along with the mean ± S.D. of the medians. Statistical analyses were performed by two-way ANOVA with comparisons to etoposide-alone treatment using Dunett’s post hoc test. (C) K562 cells incubated with the indicated concentration of MG132 or MLN7243 for 2 hours. Western blots of whole-cell extracts were probed with monoclonal antibody FK2, which recognizes all conjugated ubiquitin (monoubiquinated and polyubiquitinated proteins). ns, not significant.

Article Snippet: TOP2 covalent DNA complexes were visualized by immunofluorescence using primary antibodies for TOP2 [4566-TOP2A and 4555-TOP2B, in-house antibodies raised to the C-terminal domain of human TOP2A and TOP2B, respectively ( )] or ubiquitin (FK2, APU2, and APU3; Merck Millipore) and Alexa-488 or -594–coupled secondary antibodies (anti-rabbit A11008, or anti-mouse A11005; ThermoFisher Scientific, UK).

Techniques: Ubiquitin Proteomics, Incubation, Fluorescence, Concentration Assay, Western Blot

Journal: Molecular Pharmacology

Article Title: Small Molecule Inhibitors Confirm Ubiquitin-Dependent Removal of TOP2-DNA Covalent Complexes

doi: 10.1124/mol.119.118893

Figure Lengend Snippet: BMI1/RING1A is involved in the processing of TOP2-DNA complexes to protein-free DSBs. (A) K562 cells were treated with 100 µM etoposide (VP-16) alone or in combination with 90 µM PRT4165 for 2 hours. Cells were incubated in etoposide-free medium containing DMSO or 90 µM PRT4165 to maintain inhibition of BMI1/RING1A for 0, 0.5, 1, or 2 hours, and levels of TOP2A- and TOP2B-DNA complexes were measured using the TARDIS assay. Averages were normalized to the 2-hour continuous 100 µM etoposide control (0 hours after VP-16 removal), and statistical comparisons were made by two-way ANOVA with Bonferroni’s post hoc test (B) K562 cells were treated with 100 µM etoposide alone or in combination with 90 µM PRT4165 for 2 hours. Cells were collected and processed as per the TARDIS assay, and slides were probed with anti-ubiquitin antibody clone FK2. Averages were normalized to a 2-hour 100 µM etoposide control, and statistical comparisons were made by one-way ANOVA. (C) Cells were treated continuously with 100 µM etoposide alone or in combination with 90 µM PRT4165 for up to 4 hours, and levels of protein-free DSBs were measured using the ɣH2AX assay. Average integrated fluorescence values were normalized to the mean 1-hour 100 µM etoposide treatment value, and statistical comparisons were made by two-way ANOVA with Bonferroni’s post hoc test. Medians of independent experiments are shown as circles on the bar charts. ns, not significant.

Article Snippet: TOP2 covalent DNA complexes were visualized by immunofluorescence using primary antibodies for TOP2 [4566-TOP2A and 4555-TOP2B, in-house antibodies raised to the C-terminal domain of human TOP2A and TOP2B, respectively ( )] or ubiquitin (FK2, APU2, and APU3; Merck Millipore) and Alexa-488 or -594–coupled secondary antibodies (anti-rabbit A11008, or anti-mouse A11005; ThermoFisher Scientific, UK).

Techniques: Incubation, Inhibition, Control, Ubiquitin Proteomics, Fluorescence

Journal: Molecular Pharmacology

Article Title: Small Molecule Inhibitors Confirm Ubiquitin-Dependent Removal of TOP2-DNA Covalent Complexes

doi: 10.1124/mol.119.118893

Figure Lengend Snippet: MLN7243 inhibits the ubiquitination of TOP2-DNA complexes. (A) K562 cells transfected with control siRNA, UAE1, and UBA6 siRNA or no siRNA were treated with 0.1% DMSO or 10 µM MG132 for 2 hours. Cells without siRNA were also treated with 10 µM MLN7243 to compare levels of ubiquitination activity with UAE1/UBA6 knockdown cells. Total ubiquitination levels were examined by Western blot and probing with clone FK2 antibody, which detects all conjugated ubiquitin. (B) K562 cells were treated with 100 µM etoposide alone or in combination with 10 µM of the UAE inhibitor MLN7243 for 2 hours, and levels of ubiquitinated TOP2-DNA complexes were measured using the TARDIS assay. (C) Control cells (CON siRNA) and double–UAE1/UBA6 siRNA knockdown cells were treated with 100 µM etoposide for 2 hours, and levels of ubiquitinated TOP2-DNA complexes were quantified using the TARDIS assay ( n = 3); medians of independent experiments are shown as circles on the bar charts. Statistics was done using two-way ANOVA. Significance comparisons were made by by t test (B) or one-way ANOVA with Tukey’s post hoc test (C).

Article Snippet: TOP2 covalent DNA complexes were visualized by immunofluorescence using primary antibodies for TOP2 [4566-TOP2A and 4555-TOP2B, in-house antibodies raised to the C-terminal domain of human TOP2A and TOP2B, respectively ( )] or ubiquitin (FK2, APU2, and APU3; Merck Millipore) and Alexa-488 or -594–coupled secondary antibodies (anti-rabbit A11008, or anti-mouse A11005; ThermoFisher Scientific, UK).

Techniques: Ubiquitin Proteomics, Transfection, Control, Activity Assay, Knockdown, Western Blot

Journal: Molecular Pharmacology

Article Title: Small Molecule Inhibitors Confirm Ubiquitin-Dependent Removal of TOP2-DNA Covalent Complexes

doi: 10.1124/mol.119.118893

Figure Lengend Snippet: The effect of siRNA-mediated depletion of UAE1 on the processing of etoposide-induced TOP2-DNA complexes. (A) K562 cells were treated with UAE1 siRNA for 72 hours and then incubated for 2 hours with 100 µM VP-16 or 0.2% DMSO, which was followed by etoposide removal and a further 2 hours incubation in etoposide-free medium. Cells were collected at 0, 0.5, 1, and 2 hours after etoposide removal, and TOP2-DNA complex levels were quantified by TARDIS assay. Statistical comparisons were made by two-way ANOVA with Bonferroni’s post hoc test. (B) siRNA knockdown of UAE1 for 24, 48, 72, and 96 hours in K562 cells was tested by Western blot probing for UAE1.

Article Snippet: TOP2 covalent DNA complexes were visualized by immunofluorescence using primary antibodies for TOP2 [4566-TOP2A and 4555-TOP2B, in-house antibodies raised to the C-terminal domain of human TOP2A and TOP2B, respectively ( )] or ubiquitin (FK2, APU2, and APU3; Merck Millipore) and Alexa-488 or -594–coupled secondary antibodies (anti-rabbit A11008, or anti-mouse A11005; ThermoFisher Scientific, UK).

Techniques: Incubation, Knockdown, Western Blot

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes

doi: 10.3390/ijms241512010

Figure Lengend Snippet: TSC ligand inhibition on Tops.

Article Snippet: Ni(L1)(H1)Cl, Ni(HL2) 2 Cl 2 , Ni(L3) 2 ,Ni(L4) 2 Ni(L5) 2 Cl 2 , No inhibition of Top2 (TopoGEN) , [ ] .

Techniques: Inhibition, Labeling, Isolation

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes

doi: 10.3390/ijms241512010

Figure Lengend Snippet: Metal–TSC inhibition of Top2.

Article Snippet: Ni(L1)(H1)Cl, Ni(HL2) 2 Cl 2 , Ni(L3) 2 ,Ni(L4) 2 Ni(L5) 2 Cl 2 , No inhibition of Top2 (TopoGEN) , [ ] .

Techniques: Inhibition, Isolation, Ligation

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes

doi: 10.3390/ijms241512010

Figure Lengend Snippet: Examples of metal–TSC complexes studied with Top2. Several metal–TSC complexes are shown along with some varying side-chain examples.

Article Snippet: Ni(L1)(H1)Cl, Ni(HL2) 2 Cl 2 , Ni(L3) 2 ,Ni(L4) 2 Ni(L5) 2 Cl 2 , No inhibition of Top2 (TopoGEN) , [ ] .

Techniques:

Journal: International Journal of Molecular Sciences

Article Title: Inhibition of Topoisomerases by Metal Thiosemicarbazone Complexes

doi: 10.3390/ijms241512010

Figure Lengend Snippet: Mechanisms of TSCs against Top1B (Type IB) and Top2α/β (Type IIA). Metal–TSCs appear to inhibit plasmid DNA relaxation and ATP hydrolysis. Some metal TSCs have been shown to increase plasmid DNA cleavage and/or inhibit religation.

Article Snippet: Ni(L1)(H1)Cl, Ni(HL2) 2 Cl 2 , Ni(L3) 2 ,Ni(L4) 2 Ni(L5) 2 Cl 2 , No inhibition of Top2 (TopoGEN) , [ ] .

Techniques: Plasmid Preparation