primary antibodies directed against top2a  (Novus Biologicals)

Journal: Nucleic Acids Research

Article Title: Nonredundant roles of topoisomerase 2α and 2β in the cytosolic replication of vaccinia virus

doi: 10.1093/nar/gkaf566

Figure Lengend Snippet: Cytosolic translocation of TOP2A and TOP2B during vaccinia infection. ( A ) Immunofluorescence analysis of HeLa cells infected with vaccinia virus for the indicated times demonstrates that endogenous TOP2A and TOP2B are recruited to cytosolic viral DNA early during infection (white arrows). ( B ) TOP2A and TOP2B translocation to the cytosol is dependent on early gene expression (CHX treatment) but not viral DNA replication and factory formation (AraC treatment). ( C ) Immunoblot analysis reveals that knockdown of vaccinia uncoating factor D5 leads to loss of post-replicative (F13) but not early (I3) viral proteins. Vinculin represents the cell loading control. ( D ) Loss of D5 does not impair the cytosolic translocation of TOP2A and TOP2B, as both isoforms show diffuse cytosolic staining. ( E ) Knockdown of the viral ligase A50 prevents the recruitment of TOP2A and TOP2B to viral factories (white arrows) but does not affect their cytosolic translocation. Scale bars represent 10 μm.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12 286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), double-stranded RNA (dsRNA) (RNT-SCI-10010200, Scicons, 1:1000 dilution), I3 [ ] (1:1000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Translocation Assay, Infection, Immunofluorescence, Virus, Gene Expression, Western Blot, Knockdown, Control, Staining

Journal: Nucleic Acids Research

Article Title: Nonredundant roles of topoisomerase 2α and 2β in the cytosolic replication of vaccinia virus

doi: 10.1093/nar/gkaf566

Figure Lengend Snippet: TOP2A and TOP2B interact with viral ligase A50 via their CTDs. ( A ) Schematic representation of the GFP-tagged truncation mutants analyzed in panel ( B ). (B) Confocal images showing the localization of the indicated GFP-tagged TOP2A and TOP2B mutants in live infected HeLa cells at 6 hpi. White arrows indicate cytosolic viral DNA. ( C ) Immunoblot analysis of GFP Trap pulldowns reveals that the TOP2A and TOP2B CTDs interact with Flag-tagged A50. * indicates a proteolytic product of TOP2B CTD NLS*-GFP. Scale bar represents 10 μm.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12 286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), double-stranded RNA (dsRNA) (RNT-SCI-10010200, Scicons, 1:1000 dilution), I3 [ ] (1:1000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Infection, Western Blot

Journal: Nucleic Acids Research

Article Title: Nonredundant roles of topoisomerase 2α and 2β in the cytosolic replication of vaccinia virus

doi: 10.1093/nar/gkaf566

Figure Lengend Snippet: Opposing effects of TOP2A and TOP2B knockdowns on vaccinia replication. ( A ) Immunoblot analysis confirming siRNA-mediated knockdown of TOP2A and TOP2B. ( B ) Viral DNA production at 5 h post-infection (hpi) is decreased by TOP2A knockdown and increased by TOP2B depletion. NT indicates nontargeting control siRNA. ( C ) The steady-state levels of intermediate (L4) and late (F13 and A27) viral transcripts are reduced and increased in TOP2A- and TOP2B depleted cells, respectively. ( D ) Immunoblot analysis reveals that loss of TOP2A and TOP2B results in reduced or increased expression of post-replicative viral proteins (L4, F13, and A27), respectively. ( E ) Immunofluorecence analysis reveals assembly of new virions at 8 h post infection is severely impaired in TOP2A-depleted cells but unaffected by the loss of TOP2B. RFP-A3 is a marker for virion cores (total virus) and an antibody against B5 was used to detect extracellular virions associated with the plasma membrane during viral egress. Scale bar represents 10 μm. ( F ) Quantification of extracellular virions (B5 signal, as shown in panel E) reveals a significant impairment in viral assembly and maturation in the absence of TOP2A but not TOP2B. ( G ) Quantification of plaque-forming units (PFU) in cells depleted for TOP2A and/or TOP2B show a significantly impaired production of infectious virions in TOP2A- but not TOP2B-depleted cells. All data in the bar chats are presented as mean ± SEM from at least three biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test for comparisons between treatment groups and the NT control. ns = not significant, * P ≤ 0.05, ** P ≤ 0.01, *** P ≤ 0.001, **** P ≤ 0.0001.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12 286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), double-stranded RNA (dsRNA) (RNT-SCI-10010200, Scicons, 1:1000 dilution), I3 [ ] (1:1000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Western Blot, Knockdown, Infection, Control, Expressing, Marker, Virus, Clinical Proteomics, Membrane

Journal: Nucleic Acids Research

Article Title: Nonredundant roles of topoisomerase 2α and 2β in the cytosolic replication of vaccinia virus

doi: 10.1093/nar/gkaf566

Figure Lengend Snippet: Identification of TOP2A and TOP2B interaction partners during infection. ( A ) The volcano plot shows proteins co-immunoprecipitating with TOP2A CTD NLS*-GFP or TOP2B CTD NLS*-GFP during infection identified by LC-MS in three technical replicates. Proteins significantly enriched (FDR ≤ 0.05, at least two-fold) in TOP2A CTD or TOP2B CTD samples are shown in the upper left and upper right quadrants, respectively. Host and viral proteins are represented by black and magenta dots, respectively. ( B ) Immunoblot analysis of GFP-Trap pulldowns on infected cell lysates demonstrates that TOP2A CTD NLS*-GFP interacts with viral DNA replication proteins E9 (DNA polymerase), H5 (scaffold protein), and I3 (single-stranded DNA-binding protein). In contrast, the TOP2B CTD NLS*-GFP interacts with components of the tRNA splicing ligase complex DDX1, RTCB, FAM98A, and C14orf166. A20, a component of the vaccinia DNA polymerase complex, served as a positive control and GFP alone represents a negative control. The GFP blot exposure was optimized for the Trap fractions. A longer exposure, optimized to show the input signals is shown in .

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12 286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), double-stranded RNA (dsRNA) (RNT-SCI-10010200, Scicons, 1:1000 dilution), I3 [ ] (1:1000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot, Binding Assay, Positive Control, Negative Control

Journal: Nucleic Acids Research

Article Title: Nonredundant roles of topoisomerase 2α and 2β in the cytosolic replication of vaccinia virus

doi: 10.1093/nar/gkaf566

Figure Lengend Snippet: TOP2B promotes dsRNA production and antiviral stress granule formation. ( A ) Immunofluoresce images highlighting the presence of G3BP1 positive AVGs (white arrows) in vaccinia infected cells in the presence or absence of TOP2B and E3. Scale bar represents 10 μm. ( B ) Quantification of the number of AVGs positive infected cells in the presence or absence of TOP2B and E3. Data represent mean ± SEM of counts from six biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test for comparisons between treatment groups and the NT control; **** P ≤ 0.0001. ( C ) Immunoblot analysis reveals that loss of TOP2B and E3 results in increased expression of late viral proteins. Vinculin represents the cell loading control. ( D ) Slot blot analysis of dsRNA production in infected cells treated with TOP2B, TOP2A, and nontargeting (NT) control siRNA (left) together with quantification of the levels of dsRNA (right). The data are from three biological replicates and error bars represent SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test. ns = not significant, ** P ≤ 0.01, **** P ≤ 0.0001.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12 286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), double-stranded RNA (dsRNA) (RNT-SCI-10010200, Scicons, 1:1000 dilution), I3 [ ] (1:1000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Infection, Control, Western Blot, Expressing, Dot Blot

Journal: Cardio-oncology

Article Title: Prevention of Heart Failure Induced by Doxorubicin with Early Administration of Dexrazoxane (PHOENIX Study): dose response and time course of dexrazoxane-induced degradation of topoisomerase 2b

doi: 10.1186/s40959-025-00339-0

Figure Lengend Snippet: Expression of Top2a (blue) and Top2b (orange) in PBMCs at various time points following 500 mg/m 2 dexrazoxane infusion. HR/Pre for each time point is the mean of five participants

Article Snippet: Western blot analysis was performed using antibodies against Top2a (Proteintech 20233–1-AP), Top2b (Abcam ab72334) and Lamin B1 (Abcam ab16048), followed by Peroxidase AffiniPure Goat Anti-Rabbit IgG (H + L) (Jackson ImmunoResearch Inc., 111–035-003).

Techniques: Expressing

Journal: bioRxiv

Article Title: Non-Redundant Roles of Topoisomerase 2α and 2β in the Cytosolic Replication of Vaccinia Virus

doi: 10.1101/2025.02.05.636656

Figure Lengend Snippet: ( A ) Immunofluorescence analysis of HeLa cells infected with vaccinia virus for the indicated times demonstrates that endogenous TOP2A and TOP2B are recruited to cytosolic viral DNA early during infection (white arrows). ( B ) TOP2A and TOP2B translocation to the cytosol is dependent on early gene expression (Cycloheximide treatment) but not viral DNA factory formation (AraC treatment). ( C ) Immunoblot analysis reveals that knockdown of D5 leads to loss of expression of late (F13) but not early (I3) viral proteins. Vinculin represents the cell loading control. ( D ) Loss of D5 does not impair the cytosolic translocation of TOP2A and TOP2B. ( E ) A50 knockdown prevents the recruitment of TOP2A and TOP2B to viral factories (white arrows) but does not affect their cytosolic translocation. Scale bars represent 10 µm.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1,000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1,000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), dsRNA (RNT-SCI-10010200, Scicons, 1:1,000 dilution), I3 ( ) (1:1,000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Immunofluorescence, Infection, Virus, Translocation Assay, Expressing, Western Blot, Knockdown, Control

Journal: bioRxiv

Article Title: Non-Redundant Roles of Topoisomerase 2α and 2β in the Cytosolic Replication of Vaccinia Virus

doi: 10.1101/2025.02.05.636656

Figure Lengend Snippet: ( A ) Schematic representation of the GFP-tagged truncation mutants analysed in (B). ( B ) Confocal images showing the localisation of the indicated GFP-tagged TOP2A and TOP2B mutants in live infected HeLa cells. White arrows indicate cytosolic viral DNA. ( C ) Immunoblot analysis of GFP Trap pulldowns reveals that the TOP2A and TOP2B CTDs interact with Flag-tagged A50. Scale bars represent 10 µm.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1,000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1,000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), dsRNA (RNT-SCI-10010200, Scicons, 1:1,000 dilution), I3 ( ) (1:1,000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Infection, Western Blot

Journal: bioRxiv

Article Title: Non-Redundant Roles of Topoisomerase 2α and 2β in the Cytosolic Replication of Vaccinia Virus

doi: 10.1101/2025.02.05.636656

Figure Lengend Snippet: ( A ) Immunoblot analysis of TOP2A and TOP2B siRNA mediated knockdown. ( B ) Loss of TOP2A and TOP2B results in the reduction and increase in viral DNA production at 5 hpi, respectively. NT represents the non-targeting control siRNA. ( C ) The steady-state levels of intermediate (L4) and late (F13, and A27) viral transcripts are reduced and increased in TOP2A- and TOP2B depleted cells, respectively. ( D ) Immunoblot analysis reveals that loss of TOP2A and TOP2B results in reduced or increased expression of post-replicative viral proteins (L4, F13, and A27), respectively. ( E ) Immunofluorecence analysis reevals assembly of new virions at 8 hours post infection is severely impaired in TOP2A-depleted cells but unaffected by the loss of TOP2B. RFP-A3 is a marker for virion cores (total virus) while extracellular virions associated with the plasma membrane during viral egress were detected using the B5 antibody. Scale bar represent 10 µm. ( F ) Quantification of plaque-forming units (PFU) reveals the production of infectious virions is significantly impaired in TOP2A-but not TOP2B depleted cells. All data in the bar chats are presented as mean ± SEM from at least three biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test for comparisons between treatment groups and the NT control. n.s. = not significant, *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1,000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1,000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), dsRNA (RNT-SCI-10010200, Scicons, 1:1,000 dilution), I3 ( ) (1:1,000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Western Blot, Knockdown, Control, Expressing, Infection, Marker, Virus, Membrane

Journal: bioRxiv

Article Title: Non-Redundant Roles of Topoisomerase 2α and 2β in the Cytosolic Replication of Vaccinia Virus

doi: 10.1101/2025.02.05.636656

Figure Lengend Snippet: ( A ) The volcano plot shows proteins co-immunoprecipitating with TOP2A CTD NLS*-GFP or TOP2B CTD NLS*-GFP during infection identified by LC-MS in three technical replicates. Proteins significantly enriched (FDR≤0.05, at least two-fold) in TOP2A CTD or TOP2B CTD samples are shown in the upper left and upper right quadrants, respectively. Host and viral proteins are represented black and magenta dots respectively. ( B ) Immunoblot analysis of GFP-Trap pulldowns on infected cell lysates demonstrates that TOP2A CTD NLS*-GFP interacts with viral DNA replication proteins E9 (DNA polymerase), H5 (scaffold protein) and I3 (single-stranded DNA binding protein). In contrast, the TOP2B CTD NLS*-GFP interacts with components of the tRNA splicing ligase complex DDX1, RTCB, FAM98A, and C14orf166. A20, a component of the vaccinia DNA polymerase complex, served as a positive control and GFP alone represents a negative control.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1,000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1,000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), dsRNA (RNT-SCI-10010200, Scicons, 1:1,000 dilution), I3 ( ) (1:1,000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Infection, Liquid Chromatography with Mass Spectroscopy, Western Blot, Binding Assay, Positive Control, Negative Control

Journal: bioRxiv

Article Title: Non-Redundant Roles of Topoisomerase 2α and 2β in the Cytosolic Replication of Vaccinia Virus

doi: 10.1101/2025.02.05.636656

Figure Lengend Snippet: ( A ) Immunofluoresce images highlighting the presence of G3BP1 positive AVGs (white arrows) in vaccinia infected cells in the presence or absence of TOP2B and E3. Scale bar represent 10µm. (B) Quantification of the number of AVGs positive infected cells in the presence or absence of TOP2B and E3. Data represent mean ± SEM of counts from six biological replicates. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test for comparisons between treatment groups and the NT control; ****p ≤ 0.0001. ( C ) Immunoblot analysis reveals that loss of TOP2B and E3 results in increased expression of late viral proteins. Vinculin represents the cell loading control. ( D ) Southwestern blot analysis of dsRNA production in infected cells treated with TOP2B, TOP2A and non-targeting (NT) control siRNA (left) together with quantification of the levels of dsRNA (right). The data are from three biological replicates and error bars represent SEM. Statistical significance was determined using one-way ANOVA followed by Dunnett’s post-hoc test. n.s. = not significant, **p ≤ 0.01, ****p ≤ 0.0001.

Article Snippet: Cells were stained with primary antibodies against TOP2A (Cell Signaling Technology, 12286, 1:250 dilution), TOP2B (Santa Cruz, sc-25330, 1:250), DDX1 (Proteintech, 11357-1-AP, 1:1,000 dilution), RTCB (Proteintech, 19809-1-AP, 1:50 dilution), G3BP1 (Proteintech, 66486-1-Ig, 1:1,000 dilution), FAM98A (Abcam, ab204083, 1:50 dilution), dsRNA (RNT-SCI-10010200, Scicons, 1:1,000 dilution), I3 ( ) (1:1,000 dilution), followed by Alexa Fluor 488 or 568 conjugated secondary antibodies (Invitrogen; 1:500 in blocking buffer).

Techniques: Infection, Control, Western Blot, Expressing, Southwestern Blot

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: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: TOP2A is associated with bone metastasis of liver hepatocellular carcinoma. ( A ) Volcano plot of differentially expressed genes (DEGs) between LIHC and paracancerous tissues from GSE27635 database, red indicated up-regulated genes, and blue indicated down-regulated genes. ( B ) Volcano plot of DEGs between LIHC with BM and NBM. ( C ) Heatmap of DEGs between LIHC with and without BM. ( D ) Venn diagram of DEGs from A and B. ( E ) GO and KEGG enrichment analysis of the above overlapping genes. P, peritumor; T, tumor; BM, bone metastasis; NBM, non-bone metastasis.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques:

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: The expression of TOP2A in LIHC. ( A ) The mRNA expression level of TOP2A of LIHC and normal tissues in TCGA database. ( B ) TOP2A mRNA expression of matched LIHC and normal tissues in TCGA database. ( C ) The mRNA expression level of TOP2A of LIHC and normal tissues in TCGA and GTEx database. ( D ) IHC of TOP2A protein expression in LIHC with different stage and normal liver tissues from tissue microarrays. ( E ) The mRNA expression level of TOP2A of LIHC, LIHC-BM and normal liver tissues in Tianjin Medical University Cancer Institute and Hospital. ( F ) IHC of TOP2A protein expression in three paired LIHC-BM lesions and counterpart primary LIHC tissues from Tianjin Medical University Cancer Institute and Hospital. Magnification: 200 ×, scale bars: 100 μm. *** P < 0.001.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: TOP2A expression in HCC and adjacent tissues from 10 GEO datasets.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: The prognostic value of TOP2A in LIHC. ( A ) The overall survival curve of TOP2A in LIHC. ( B ) ROC curves of TOP2A to predict the sensitivity and specificity of 1-, 2-, and 3-year overall survival. ( C ) The mRNA expression level of TOP2A of LIHC and normal tissues in ICGC database. ( D ) Distribution of risk score, survival status, and TOP2A expression profile of LIHC in ICGC database. ( E ) Kaplan-Meier curve of overall survival in high-and low-risk groups. ( F ) ROC curve of the TOP2A risk score model. HR, hazard ratio. *** P < 0.001.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: A multivariable Cox proportional hazard model for LIHC using TIMER web server.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques:

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: DNA methylation alterations of TOP2A and gene mutation analysis associated TOP2A in LIHC from TCGA database. ( A ) The methylation level of TOP2A in LIHC. ( B ) Spearman correlation between DNA methylation and mRNA expression of TOP2A in LIHC. ( C ) Progress-free survival (PFS) curve of TOP2A methylation in LIHC. ( D ) Gene mutations associated with TOP2A in LIHC. ( E ) TOP2A expression level in different TP53 mutant status in LIHC. ( F ) Overall survival (OS) curve of TP53 mutation in LIHC. *** P < 0.001.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: DNA Methylation Assay, Mutagenesis, Methylation, Expressing

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: Functional enrichment analysis of TOP2A in LIHC. ( A ) Heatmap of TOP2A co-expressed genes in LIHC using TCGA database. ( B ) KEGG and GO enrichment analysis for the up-regulated genes in LIHC. ( C ) Gene Set Enrichment Analysis (GSEA) for TOP2A in LIHC.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Functional Assay

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: Correlation analysis between TOP2A expression and pathways and markers related to metastasis or proliferation using TCGA database. ( A – C ) Spearman correlation analysis between TOP2A and PI3K-AKT-mTOR pathway, EMT markers, and tumor proliferation signature. ( D ) Spearman correlation analysis between the expression level of TOP2A and VEGFA, MMP2, and MMP9.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: Correlation analysis of TOP2A expression with immune infiltration and tumor microenvironment in LIHC. ( A ) TOP2A mRNA expression in different immune subtypes in LIHC via TISIDB. ( B ) Radar map of correlation between TOP2A expression and the abundance of immune cells. ( C ) Correlation between TOP2A expression and ESTIMATE score, Immune score, and Stromal score.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Expressing

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: Association analysis of TOP2A expression with immunotherapy in LIHC. ( A ) 8 immune checkpoint genes associated with TOP2A expression. ( B ) Radar map of correlation between TOP2A expression and immune marker sets in LIHC. ( C ) Comparison of TIDE score between the higher (G1) and lower TOP2A expression (G2) groups of LIHC. ( D ) TOP2A expression in groups with a different ICB immunotherapy response status. ** P < 0.01, *** P < 0.001.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Expressing, Marker, Comparison

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: Hippo-YAP signal pathway mediates TOP2A-driven LIHC cell growth, migration, and invasion. ( A ) The expression of TOP2A was detected by qRT-PCR and Western blot in normal liver cell line and four liver cancer cell lines. ( B ) The knockdown efficiency of TOP2A siRNA was determined by qRT-PCR and Western blot in HCCLM3 and Hep3B cells. ( C ) The expression level of p-YAP and YAP was detected by Western blot in the indicated groups of HCCLM3 and Hep3B cells. β-actin was used as a control. ( D , E ) the effect of TOP2A on cell proliferation was assessed by colony formation and CCK8 assays in HCCLM3 and Hep3B cells. ( F ) The effect of the TOP2A on cell migration and invasion was tested by transwell assays in HCCLM3 and Hep3B cells. * P < 0.05, *** P < 0.001, and **** P < 0.0001. Data presented as mean ± standard deviation (SD). Experiments were repeated at least three times.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Migration, Expressing, Quantitative RT-PCR, Western Blot, Standard Deviation

Journal: Aging (Albany NY)

Article Title: Identification of topoisomerase 2A as a novel bone metastasis-related gene in liver hepatocellular carcinoma

doi: 10.18632/aging.205216

Figure Lengend Snippet: TOP2A enhances bone-specific metastatic potential and tumor-induced osteolysis in the bone microenvironment. ( A ) Chemotactic migration of HCCLM3 and Hep3B cells toward MG63 cells was evaluated by transwell assays. ( B ) The adhesion ability of HCCLM3 and Hep3B cells to MG63 cells was assessed by heterogeneous cell-cell adhesion assay. ( C ) TRAP staining of bone marrow monocyte (BMM) cells treated with conditioned medium (CM) from HCCLM3 and Hep3B cells. Scale bar, 200 μM. Quantification of TRAP+ osteoclast number. ( D ) qRT-PCR verified the expression of TRAP, CTSK, and NFACT1 in the indicated groups of BMM cells. GAPDH as the loading control. ( E ) 2.5×10 5 control or TOP2A knockdown HCCLM3 and Hep3B cells were injected directly into the tibias of the mice. Representative bioluminescent images taken on day 28 after inoculation are shown. ( F ) Representative micro-CT images of tibia osteolytic lesions. ( G ) H&E and TRAP staining of tibia lesions of mice. Quantification of TRAP+ osteoclasts. Scale bar, 100 μm. * P < 0.05, ** P < 0.01. B, bone; M, bone marrow; T, tumor. Data presented as mean ± SD. Experiments were repeated at least three times.

Article Snippet: Protein samples were separated using 10% SDS-PAGE for 2 h and transferred into PVDF membranes at 250 mA for 1.5 h. After blocking with 10% non-fat milk for 2 h, primary antibodies against TOP2A (dilution 1:1000; cat no.ab52934, Abcam, USA), YAP (dilution 1:1000; cat no.ab52771, Abcam, USA), p-YAP (dilution 1:1000; cat no. ab76252, Abcam, USA) and β-actin (dilution 1:12000; cat no. AF7018, Affinity, China) were incubated at 4° C overnight.

Techniques: Migration, Cell Adhesion Assay, Staining, Quantitative RT-PCR, Expressing, Injection, Micro-CT