Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: siRNAs and their target genes used in the study

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Sequencing

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: TOP2A is involved in drug resistance to regorafenib. (A) Kaplan-Meier curves for HCC patients with low vs. high expression of TOP2A using TCGA database. (B, C) TOP2A expression in sorafenib-resistant cell lines and corresponding parental cell lines detected by Western blot and normalized to β-actinin (B); or quantitative reverse transcription-polymerase chain reaction (qRT-PCR) and normalized to glyceraldehyde 3-phosphate dehydrogenase (GAPDH) (C). (D) Huh7 and HepG2 cells were incubated in gradually increased concentrations of sorafenib for 48 h. Expression of TOP2A was detected by Western blot. Density of each band was normalized to that of β-actin. (E-H) Sorafenib-resistant Huh7-SR and HepG2-SR cells and corresponding parental cells were incubated with 0, 2.5, 5, 7.5 μM regorafenib for 48 h. The protein expression profiles were detected by Western blot. Density of each band was normalized to that of β-actin (E, F). Sorafenib-resistant HCC cells and parental cells were incubated with gradually increased concentrations of regorafenib or sorafenib for 48 h. TOP2A mRNA levels were measured by qRT-PCR and normalized against GAPDH. The relative TOP2A mRNA levels of cells treated with 0 μM sorafenib or regorafenib were normalized to 1 (G, H). *P < 0.05; **P < 0.01; and ***P < 0.001 vs. the sorafenib or regorafenib untreated cells.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Expressing, Western Blot, Reverse Transcription, Polymerase Chain Reaction, Quantitative RT-PCR, Incubation

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: TOP2A silencing enhances sensitivity to regorafenib in sorafenib-resistant HCC cells. A, B. Sorafenib-resistant Huh7-SR and HepG2-SR cells were transfected with siTOP2A or negative control (NC) for 48 h. The corresponding un-transfected cells served as control. The protein expression profiles were detected by Western blot. Density of each band was normalized to that of β-actin. C, D. Huh7-SR and HepG2-SR cells and corresponding parental cells were transfected with siTOP2A for 24 h; subsequently incubated with increasing concentrations of regorafenib (Reg) for 48 h. Viability of transfected cells was normalized to control. E, F. Sorafenib-resistant cells and parental cells were exposed to 7.5 μM regorafenib for different periods, after transfected with siTOP2A. Cell viability was normalized with corresponding control. G, H. Huh7-SR and HepG2-SR cells were transfected with siTOP2A for 24 h; subsequently incubated with 7.5 μM regorafenib for 48 h. Protein expression profiles were detected by Western blot. Density of each band was normalized to that of β-actin. *P < 0.05; **P < 0.01 compared siTOP2A or regorafenib to control. #P < 0.05; ##P < 0.01 compared with regorafenib treated cells. †P < 0.05; ††P < 0.01 compared with siTOP2A treated cells.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Transfection, Negative Control, Control, Expressing, Western Blot, Incubation

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: Inhibition of TOP2A synergizes with regorafenib to suppress cell viability, promote apoptosis, and strengthen sensitivity of sorafenib-resistant HCC cells to regorafenib in vitro. (A-D) HepG2-SR and Huh7-SR as well as corresponding parental cells were exposed to different concentrations of doxorubicin (Dox) or/and regorafenib (Reg) for 48 h (A, B); or incubated for different periods in the presence or absence of regorafenib (7.5 μM) and doxorubicin (160 nM) (C, D). Cell viability (%) was compared with control. (E) Huh7-SR and HepG2-SR cells were incubated with 7.5 μM regorafenib or/and 160 nM doxorubicin for 48 h. The protein expression profile of cyclin D1 and cleaved caspase-3 was detected by Western blot. Density of each band was normalized to that of β-actin. (F) Migration of sorafenib-resistant HCC cells was examined by Wound healing assay (100 ×). (G) Transwell assays to examine synergistic effects of doxorubicin with regorafenib on migration and invasion (100 ×). (H) Synergistic effects of doxorubicin and regorafenib on 3D invasion (40 ×). *P < 0.05; **P < 0.01; indicating a significant difference from untreated cells. #P < 0.05; ##P < 0.01 compared with regorafenib treatment. †P < 0.05; ††P < 0.01 compared with doxorubicin treated cells.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Inhibition, In Vitro, Incubation, Control, Expressing, Western Blot, Migration, Wound Healing Assay

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: Inhibition of TOP2A synergizes with regorafenib to enhance the antitumor activity of regorafenib in vivo. (A-C) Subcutaneous xenografts were established. Mice received different treatments for 15 days. (A) Tumor images. (B) Tumor volume (mm3) was recorded. (C) Tumors were harvested and weighed. (D, E) Expression of cyclin D1 and cleaved caspase-3 by Western blot. Density of each band was normalized to that of β-actin (E). (F-I) Hematoxylin and eosin (H&E) (magnification, upper × 100, lower × 400) and immunohistochemistry staining (magnification, × 200). *P < 0.05; and **P < 0.01. #P < 0.05; ##P < 0.01 compared with regorafenib group. †P < 0.05; ††P < 0.01 compared with doxorubicin treated group.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques: Inhibition, Activity Assay, In Vivo, Expressing, Western Blot, Immunohistochemistry, Staining

Journal: American Journal of Cancer Research

Article Title: TOP2A inhibition reverses drug resistance of hepatocellular carcinoma to regorafenib

doi:

Figure Lengend Snippet: Schematic diagram depicting hypothesized roles of TOP2A in acquired resistance to regorafenib in HCC. →, positive regulation; ⊥, negative regulation or blockade.

Article Snippet: The antibodies (Abs) against TOP2A and cyclin D1 were purchased from ABclonal Biotech Co., Ltd. (Wuhan, China).

Techniques:

Journal: Oncotarget

Article Title: Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity

doi: 10.18632/oncotarget.23543

Figure Lengend Snippet: ( A ) Heat map for the changes of gene expression in human primary cardiomyocytes treated with trastuzumab, trastuzumab plus pertuzumab or T-DM1. Data with opposite influence on gene expression among these three treatments were removed, and then were further selected with threshold +/– 1.2 fold changes and P < 0.05 as selection criteria. This results in 2383 genes, which were input into the website Morpheus to generate a heat map according to the instruction. Following parameters were used to generate the heatmap with hierarchical clustering (metric: euclidean distance; linkage method: average). ( B ) Number of genes either up- or down- regulated by trastuzumab, trastuzumab plus pertuzumab or T-DM1. ( C ) Gene expression changes in DNA topoisomerase IIA (TOP2A) and IIB (TOP2B) in human primary cardiomyocytes treated by T-DM1, trastuzumab, or trastuzumab plus pertuzumab. ( D ) Gene clusters affected by trastuzumab treatment in human primary cardiomyocyte.

Article Snippet: Antibody against TOP2A was obtained from Origene.

Techniques: Expressing, Selection

Journal: Oncotarget

Article Title: Type IIB DNA topoisomerase is downregulated by trastuzumab and doxorubicin to synergize cardiotoxicity

doi: 10.18632/oncotarget.23543

Figure Lengend Snippet: ( A ) Human primary cardiomyocytes were treated with trastuzumab, pertuzumab, trastuzumab plus pertuzumab, ramucirumab and cetuximab at 50 µg/ml for 24 h. After treatments, Western blotting was performed, and expression levels of TOP2B and TOP2A were detected using antibodies directed against TOP2B or TOP2A. Equal loading of protein samples were confirmed by actin blots. Upper two panels: TOP2B expression; lower two panels: TOP2A expression. ( B ) TOP2B protein levels in human primary cardiomyocytes that were either treated with trastuzumab at 50 µg/ml (left graph) or doxorubicin (Dox) at 0.5 µM (right graph) or left untreated for 24 h. TOP2B expression was monitored using flow cytometry. ( C ) Quantitative analysis of fluorescent intensity obtained from flow cytometry analysis was presented in the form of bar graphs. Data are representative of two or more independent experiments and expressed as mean ± SEM. ( D ) TOP2B protein levels in human primary cardiomyocytes that were treated with trastuzumab (50 µg/ml), Dox (0.5 µM), trastuzumab (50 µg/ml) plus Dox (0.5 µM) or left untreated for 24h. TOP2B protein levels were monitored using flow cytometry. ( E ) TOP2B protein levels in human primary cardiomyocytes that were treated with Dox (0.5 µM) for 24 h or left untreated for 48 h. After 24h Dox treatment, Dox was removed from cell culture media, and cells were then either treated with trastuzumab (50 µg/ml) or left untreated for additional 24h. TOP2B protein levels were monitored using flow cytometry. The isotype in Figure and is a negative control. Experiments were performed in triplicates, and data are representative of two - three independent experiments. Student’s t -test was used for statistical significance. * P < 0.05; ** P < 0.01.

Article Snippet: Antibody against TOP2A was obtained from Origene.

Techniques: Western Blot, Expressing, Flow Cytometry, Cell Culture, Negative Control

Journal: Cell Division

Article Title: Anti-cancer effects of Coix seed extract through KCTD9-mediated ubiquitination of TOP2A in lung adenocarcinoma

doi: 10.1186/s13008-024-00112-2

Figure Lengend Snippet: KCTD9 affects the level of ubiquitination of TOP2A. a Interactions between TOP2A and TOP2B and KCTD9 in the String database. Protein expression of TOP2A ( b ) and TOP2B ( c ) in LUAD was analyzed in the UALCAN database. d Multiple ubiquitination modification sites are present in TOP2A in the GPS-Uber database. e The mRNA expression of KCTD9 in LUAD cells after infection of sh-NC or sh-KCTD9 was verified using RT-qPCR. f The mRNA expression of TOP2A in LUAD cells after infection of sh-NC or sh-KCTD9 was verified using RT-qPCR assays. g The protein expression of TOP2A in LUAD cells after infection of sh-NC or sh-KCTD9 was verified using western blot assays. The expression of TOP2A in A549 ( h ) and HCC827 ( i ) cells after infection of sh-NC or sh-KCTD9 in the presence of CHX. j Effect of KCTD9 on TOP2A ubiquitination detected by immunoprecipitation. k The protein expression of TOP2A in LUAD cells after infection of sh-NC or sh-KCTD9 with the proteasome inhibitor MG132 treatment. Data are presented as the mean ± SD of results from at least three independent experiments performed in triplicates, two-way ANOVA with Tukey’s posttest, * p < 0.05

Article Snippet: The supernatant was immunoprecipitated with protein magnetic beads, which were incubated with the antibody against TOP2A (1:200, ab12318, Abcam).

Techniques: Expressing, Modification, Infection, Quantitative RT-PCR, Western Blot, Immunoprecipitation

Journal: Cell Division

Article Title: Anti-cancer effects of Coix seed extract through KCTD9-mediated ubiquitination of TOP2A in lung adenocarcinoma

doi: 10.1186/s13008-024-00112-2

Figure Lengend Snippet: Silencing of TOP2A reverses the effects of KCTD9 knockdown on LUAD cell immune escape. a Correlation of TOP2A expression in LUAD with human CD8 + T cell infiltration predicted in the TIMER database. b Correlation between TOP2A and PD-L1 expression predicted in the GEPIA database. c The prognostic outcomes in patients with high or low TOP2A expression are predicted in the Kaplan–Meier Plotter database. d The mRNA expression of TOP2A in LUAD cells after infection of sh-NC or sh-TOP2A was determined using RT-qPCR. e The mRNA expression of PD-L1 in LUAD cells was detected using RT-qPCR. f The protein expression of PD-L1 in LUAD cells was detected using western blot assays. g - j The levels of TNF-α, IFN-γ, CXCL10, and CXCL9 in the co-culture system of LUAD cells and human CD8 + T cells. k Proliferation of human CD8 + T cells in co-culture system detected by CFSE assays. l The ability of differently treated LUAD cells against human CD8 + T cells was evaluated using tumor cell killing assay. Data are presented as the mean ± SD of results from at least three independent experiments performed in triplicates, two-way ANOVA with Tukey’s posttest, * p < 0.05

Article Snippet: The supernatant was immunoprecipitated with protein magnetic beads, which were incubated with the antibody against TOP2A (1:200, ab12318, Abcam).

Techniques: Expressing, Infection, Quantitative RT-PCR, Western Blot, Co-Culture Assay

Journal: Cell Division

Article Title: Anti-cancer effects of Coix seed extract through KCTD9-mediated ubiquitination of TOP2A in lung adenocarcinoma

doi: 10.1186/s13008-024-00112-2

Figure Lengend Snippet: The inhibiting effects of CSE on the immune escape of LUAD cells in vivo are reversed by KCTD9 knockdown. a Tumor volume in mice in 3 weeks. b Weight changes in mouse tumors. c The protein expression of PD-L1 in mouse tumor tissues was examined using western blot assays. d Immunofluorescence analysis of CD8 + T cell infiltration in mouse tumor tissues. e The levels of CD8 + T cell activation markers GzmB and Perforin in the supernatant of the co-culture system were detected by ELISA. f Detection of apoptosis in mouse tumor tissues by TUNEL assay. The levels of TNF-α ( g ), IFN-γ ( h ), CXCL10 ( i ), and CXCL9 ( j ) in the mouse tumor tissues by ELISA. ( k ) The mRNA expression of KCTD9 in mouse tumor tissues was assessed by RT-qPCR. l The protein expression of TOP2A in mouse tumor tissues was assessed by western blot assays. Data are presented as the mean ± SD of results from at least three independent experiments performed in triplicates, one-way or two-way ANOVA with Tukey’s posttest, * p < 0.05

Article Snippet: The supernatant was immunoprecipitated with protein magnetic beads, which were incubated with the antibody against TOP2A (1:200, ab12318, Abcam).

Techniques: In Vivo, Expressing, Western Blot, Immunofluorescence, Activation Assay, Co-Culture Assay, Enzyme-linked Immunosorbent Assay, TUNEL Assay, Quantitative RT-PCR

Journal: Cell Death & Disease

Article Title: Pericentromeric Satellite III transcripts induce etoposide resistance

doi: 10.1038/s41419-021-03810-9

Figure Lengend Snippet: A Representative images of the SatIII RNA and TOP2A co-localization in HeLa cells exposed to HS conditions (1 h at 44 °C) or HS plus 24 h recovery at 37 °C and DMSO or etoposide 10 µM treatment. SatIII RNA was stained using smFISH (red), TOP2A was stained using a protein-specific antibody (green). Scale bar, 10 µm. B Quantification of ( A ) by counting the number of foci per cell. Quantification was performed using an automated ImageJ pipeline, n = 5. C Cell cycle assay utilizing Hoechst staining was performed to clarify SatIII expression patterns during the cell cycle. HeLa cells were subjected to HS (1 h at 44 °C) or control conditions, fixed and stained with Hoechst staining dye. The cells were monitored over the course of the cell cycle in an automated HCS microscope. A minimum of 3000 cells, separated in n = 6 replicates, were quantified for each condition. D Effects of SatIII RNA knockdown on DNA damage was investigated by immunofluorescence staining for 53BP1. HeLa cells were transfected with siSatIII and scramble RNA (control), respectively. Cells were then exposed to HS conditions (1 h at 44 °C) or constant 37 °C and treated with 20 µM etoposide or DMSO. After 24 h, cells were fixed and stained with a protein-specific 53BP1antibody (green). Counterstaining of nuclei was performed with Hoechst stain. Imaging and analyses were performed utilizing HCS microscope-based quantifications of the staining. Error bars represent the standard deviation of the mean of three replicates. Two-tailed paired Student’s t test significant P -values are marked: < 0.05 with (*), < 0.01 with (**), < 0.001 with (***). Scale bar, 10 µm. E RNA immunoprecipitation in HeLa cells subjected to three different treatment conditions: HS (1 h at 44 °C), HS with a 24 h recovery time at 37 °C (HS + rec), and non-HS conditions (nHS, 37 °C). Chromatin was sheared by sonication and precipitated using an antibody against human TOP2A or HSF1. Binding to SatIII was analyzed using qPCR. HSF1 was used as a positive control. Figure shows a typical result for two biological replicates, each with three technical replicates. F Caspase-3/-7 assay of HeLa cells either transfected with the siRNA targeting SatIII (siSatIII) or control siRNA (siCo). After transfection, cells were treated as indicated with etoposide or a DMSO control. Error bars represent standard deviation of the mean of three replicates. Two-tailed paired Student’s t-test significant P -values are marked: < 0.05 with (*), < 0.01 with (**), < 0.001 with (***).

Article Snippet: RNA was precipitated using an antibody against human TOP2A (SigmaAldrich, #SAB4502998, RRID:AB_10753226) and HSF1 (Santa Cruz Biotechnology, #sc-17757, RRID:AB_627753).

Techniques: Staining, Cell Cycle Assay, Expressing, Microscopy, Immunofluorescence, Transfection, Imaging, Standard Deviation, Two Tailed Test, Immunoprecipitation, Sonication, Binding Assay, Positive Control

Journal: Cell Death & Disease

Article Title: Pericentromeric Satellite III transcripts induce etoposide resistance

doi: 10.1038/s41419-021-03810-9

Figure Lengend Snippet: Loss of BRCA1 results in an increased SatIII RNA expression through reduced ubiquitination of H2A and a relaxation of pericentromeric heterochromatin, reflected by a loss of H3K9me2 (left side, Zhu et al., 2018, Padeken et al.). SatIII RNA interacts with the BRCA1-associated protein network and destabilizes replication forks which in turn enhances DNA damage and genomic instability, ultimately promoting tumor growth. Etoposide also drives SatIII expression, but in this case, SatIII RNA facilitates the recruitment of TOP2A to TOP2ccs located at nSBs (our data). This leads to less DNA damage and subsequent downstream mechanisms that decrease genomic instability and therefore cells are more resistant against etoposide.

Article Snippet: RNA was precipitated using an antibody against human TOP2A (SigmaAldrich, #SAB4502998, RRID:AB_10753226) and HSF1 (Santa Cruz Biotechnology, #sc-17757, RRID:AB_627753).

Techniques: RNA Expression, Expressing

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Primers for RT-qPCR.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Sequencing

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Sequences of siRNAs.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Sequencing, Negative Control

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: TOP2A is high expressed in MB. (A) The expression of TOP2A in normal brain tissues and MB tissues was analyzed according to microarray data (GSE50161, GSE39182, GSE74195 and GSE35493). Values are the median with 95% CI. (B) The expression of TOP2A in 3 pairs of MB tissues and corresponding normal brain tissues was analyzed through RT-qPCR analysis. Transcript levels were normalized to GAPDH expression. *P<0.05. (C) The expression of TOP2A in MB tissues and adjacent normal brain tissues was detected through IHC (magnification × 200 in each picture). (D) The expression of TOP2A was assessed in 20 types of paediatric brain tumors with UALCAN, including MB (indicated by a black box). (E) The expression of TOP2A across various tumor cell lines, including MB cell lines (indicated by a red box), is shown in the CCLE database.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Expressing, Microarray, Quantitative RT-PCR

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: The relationship between the expression level of TOP2A and clinicopathological features in GSE85217 dataset. (A) The expression level of TOP2A in different genders. (B) The expression level of TOP2A in different age groups. (C) The expression level of TOP2A in different metastatic status. (D) The expression level of TOP2A in different histology types.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Prognostic analysis of TOP2A in MB patients in GSE85217 dataset. (A) Kaplan-Meier survival curves of the high and low TOP2A expression groups divided by the median score. (B) ROC curve analysis of the prognostic value of TOP2A for MB patients. (C) Nomogram for predicting 3- and 5-year survival rates of MB patients. The variables include gender (0, female; 1, male), age (1, 0-3 years; 2, 3-10 years; 3, 10-17 years; 4, 18+ years), histology (1, classic; 2, desmoplastic; 3, LCA; 4, MBEN), met status (0, M0; 1, M1), molecular subgroup (1, WNT; 2, SHH; 3, Group3; 4, Group4) and TOP2A expression, which were integrated in the nomogram. (D, E) The calibration curves for predicting survival probability at 3 and 5 years.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Expressing

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Upregulation of TOP2A promotes MB cell proliferation and invasion. (A) The expression of TOP2A was detected by western blot analysis after transfecting Daoy cells with pcDNA3.1-TOP2A. (B) Semi-quantitative densitometric analysis was used to measure the relative levels of TOP2A. **P<0.01. (C) CCK-8 assay was used to detect the proliferation ability of pcDNA3.1-TOP2A-transfected Daoy cells. **P<0.01, ***P<0.001. (D) Transwell assays precoated with Matrigel Matrix were performed to assess the invasive ability of pcDNA3.1-TOP2A-transfected Daoy cells. ***P<0.001.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Expressing, Western Blot, CCK-8 Assay, Transfection

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Downregulation of TOP2A inhibits MB cell proliferation. (A) The expression of TOP2A was detected by RT-qPCR after transfecting Daoy cells with TOP2A siRNAs. *P<0.05, **P<0.01, ***P<0.001. (B) The expression of TOP2A was detected by western blot analysis after transfecting Daoy cells with si-TOP2A. (C) Semi-quantitative densitometric analysis was used to measure the relative levels of TOP2A. **P<0.01. (D) CCK-8 assay was used to detect the proliferation ability of si-TOP2A-transfected MB cells with or without exposure to 8 Gy IR. ***P<0.001. (E) The relative proliferation of si-TOP2A-transfected MB cells was shown at days 6 and 7 post IR treatment. ***P<0.001.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Expressing, Quantitative RT-PCR, Western Blot, CCK-8 Assay, Transfection

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Downregulation of TOP2A inhibits MB cell migration and invasion. (A) Wound healing assays were performed to evaluate the migratory ability of si-TOP2A-transfected MB cells with or without exposure to 8 Gy IR. *P<0.05, **P<0.01, ***P<0.001. (B) Transwell assays were performed to assess the migratory ability of si-TOP2A-transfected MB cells with or without exposure to 8 Gy IR. **P<0.01. (C) Transwell assays precoated with Matrigel Matrix were performed to assess the invasive ability of si-TOP2A-transfected MB cells with or without exposure to 8 Gy IR. *P<0.05.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Migration, Transfection

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Downregulation of TOP2A limits the radioresistance of MB cells. (A) Colony formation assays were employed to evaluate the radiosensitivity of MB cells individually exposed to 0, 2, 4, 6, 8, and 10 Gy IR, respectively. (B) Cell survival curves analysis were performed to assess the radioresistance of MB cells.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques:

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Parameters of radiosensitivity in different groups of Daoy cells.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques:

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Downregulation of TOP2A inhibits the activity of the Wnt/β-catenin signaling pathway. (A) Western blot assay was used to detect the expression of β-catenin following TOP2A knockdown alone or combined treated with 8 Gy IR. (B) Semi-quantitative densitometric analysis was used to measure the relative levels of TOP2A and β-catenin following TOP2A knockdown alone or combined treated with 8 Gy IR. *P<0.05. (C) Western blot assay was used to detect the expression of β-catenin following TOP2A overexpression alone or combined treated with 8 Gy IR. (D) Semi-quantitative densitometric analysis was used to measure the relative levels of TOP2A and β-catenin following TOP2A overexpression alone or combined treated with 8 Gy IR. *P<0.05, **P<0.01. (E) The expression of TOP2A in cerebellum tissues and MB molecular subgroups was analyzed in GSE109401. *P<0.05, **P<0.01. (F) The expression of TOP2A in MB molecular subgroups was analyzed in GSE85217. **P<0.01, ***P<0.001.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Activity Assay, Western Blot, Expressing, Knockdown, Over Expression

Journal: Frontiers in Oncology

Article Title: TOP2A correlates with poor prognosis and affects radioresistance of medulloblastoma

doi: 10.3389/fonc.2022.918959

Figure Lengend Snippet: Enrichment pathways analysis of TOP2A based on GSE85217 dataset. (A) HALLMARK_WNT_BETA_CATENIN signaling pathways enriched across DEGs by GSEA analysis. (B) PPI network of the genes of Wnt/β-catenin signaling pathway in GSEA analysis constructed by STRING analysis. (C K) Co-expression analysis of TOP2A with the core genes of Wnt/β-catenin signaling pathway.

Article Snippet: Primary antibody against TOP2A (Proteintech Group, Chicago, IL, USA) was diluted at 1:100 and incubated with the MB sections at 4°C overnight.

Techniques: Protein-Protein interactions, Construct, Expressing

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology *

doi: 10.1074/mcp.RA119.001626

Figure Lengend Snippet: Significantly regulated proteins that are more abundant in low-quality human spermatozoa following Percoll density gradient separation

Article Snippet: Antibodies used and their dilutions were as follows: mouse monoclonal antibody against TOP2A (Abcam, ab52934) diluted 1/1000, sheep polyclonal antibody against PDIA3 (Bio-techne, AF8219) diluted 1/1000, mouse monoclonal against alpha tubulin (Sigma, T5168) diluted 1/2000, rabbit polyclonal against histone H3 (Sigma, H0164) diluted 1/3000.

Techniques:

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology *

doi: 10.1074/mcp.RA119.001626

Figure Lengend Snippet: TOP2A is more abundant in spermatozoa derived from the low quality fractions. Human spermatozoa from individual donors were fractionated through Percoll density gradients. Intact cells were lysed and run into SDS-PAGE. Following transfer, the nitrocellulose was probed with primary antibodies against A, TOP2A (upper blot). Cross-reacting bands were visualized with chemiluminescence after addition of horse-radish peroxidase conjugated secondary antibody. Lower Blot: Following recording of chemiluminescent, the nitrocellulose was stripped of primary antibody, re-blocked and probed with anti-α Tubulin. B, The TOP2A abundance according to the SWATH analysis between high- and low-quality sperm cells demonstrates significantly more abundance within the latter (n = 8, *p = 0.0001).

Article Snippet: Antibodies used and their dilutions were as follows: mouse monoclonal antibody against TOP2A (Abcam, ab52934) diluted 1/1000, sheep polyclonal antibody against PDIA3 (Bio-techne, AF8219) diluted 1/1000, mouse monoclonal against alpha tubulin (Sigma, T5168) diluted 1/2000, rabbit polyclonal against histone H3 (Sigma, H0164) diluted 1/3000.

Techniques: Derivative Assay, SDS Page

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology *

doi: 10.1074/mcp.RA119.001626

Figure Lengend Snippet: Immunofluorescent detection of TOP2A in spermatozoa demonstrates the protein is associated with amorphous heads. Spermatozoa were fixed and settled onto poly-lysine slides. Following permeabilization with 0.2% Triton-X and blocking with 3% BSA, the cells were incubated with primary anti-TOP2A antibody then secondary alexafluor-488-congucated antibody and counterstained with DAPI A, (i) Phase image of spermatozoa, (ii) Immunofluorescent image using anti-TOP2A antibody fluorescence within spermatozoa, (iii) DAPI staining of spermatozoon DNA, (iv) composite image of DAPI and TOP2A fluorescence. Scale bar = 10 μm. B, Comparison of cells with detected TOP2A fluorescence between high- and low-quality cell populations (n = 3, *p = <0.0001).

Article Snippet: Antibodies used and their dilutions were as follows: mouse monoclonal antibody against TOP2A (Abcam, ab52934) diluted 1/1000, sheep polyclonal antibody against PDIA3 (Bio-techne, AF8219) diluted 1/1000, mouse monoclonal against alpha tubulin (Sigma, T5168) diluted 1/2000, rabbit polyclonal against histone H3 (Sigma, H0164) diluted 1/3000.

Techniques: Blocking Assay, Incubation, Fluorescence, Staining

Journal: Molecular & Cellular Proteomics : MCP

Article Title: Proteomic Analysis Reveals that Topoisomerase 2A is Associated with Defective Sperm Head Morphology *

doi: 10.1074/mcp.RA119.001626

Figure Lengend Snippet: TOP2A is associated with amorphous sperm heads. Cells from either the high- or low-quality Percoll fractionation were stained immunostained for TOP2A. Those cells positive for the protein, were then assessed in terms of their morphology. The graph shows that combined cell counts for both high- and low-quality spermatozoa (n = 3 donors, 100 cells counted/fraction).

Article Snippet: Antibodies used and their dilutions were as follows: mouse monoclonal antibody against TOP2A (Abcam, ab52934) diluted 1/1000, sheep polyclonal antibody against PDIA3 (Bio-techne, AF8219) diluted 1/1000, mouse monoclonal against alpha tubulin (Sigma, T5168) diluted 1/2000, rabbit polyclonal against histone H3 (Sigma, H0164) diluted 1/3000.

Techniques: Fractionation, Staining

Journal: Journal of Translational Medicine

Article Title: Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer

doi: 10.1186/1479-5876-11-36

Figure Lengend Snippet: Immunostaining for TOP2A in prostate cancer observed at 200 magnification showing 3+ staining (A); 2+ staining (B); 1+ staining (C); and negative staining (D).

Article Snippet: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO).

Techniques: Immunostaining, Staining, Negative Staining

Journal: Journal of Translational Medicine

Article Title: Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer

doi: 10.1186/1479-5876-11-36

Figure Lengend Snippet: Examples of fluorescent in situ hybridization for TOP2A gene (orange) and chromosome 17 (green) showing no copy number alteration.

Article Snippet: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO).

Techniques: In Situ Hybridization

Journal: Journal of Translational Medicine

Article Title: Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer

doi: 10.1186/1479-5876-11-36

Figure Lengend Snippet: Biochemical recurrence-free survival curves showing lower survival rates of those cases which expressed high levels of TOP2A protein.

Article Snippet: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer

doi: 10.1186/1479-5876-11-36

Figure Lengend Snippet: Association between TOP2A expression and biochemical recurrence - free survival

Article Snippet: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO).

Techniques: Expressing

Journal: Journal of Translational Medicine

Article Title: Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer

doi: 10.1186/1479-5876-11-36

Figure Lengend Snippet: Multivariate analysis for biochemical recurrence - free survival

Article Snippet: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO).

Techniques:

Journal: Journal of Translational Medicine

Article Title: Prognostication of prostate cancer based on TOP2A protein and gene assessment: TOP2A in prostate cancer

doi: 10.1186/1479-5876-11-36

Figure Lengend Snippet: Biochemical recurrence - free survival curves concerning fractal analysis of a subset of 20 randomly selected cases showing lower survival rates of those who expressed higher levels of TOP2A protein.

Article Snippet: Immunohistochemistry (IHC) was automated using mouse monoclonal antibody against TOP2A (clone SWT3D1; DAKO, Carpenteria, CA, USA) at dilution 1:800 and Flex Plus detection system in autostainer 48Ultra (DAKO).

Techniques: