Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: (A) CRC whole cell extract (40 μg) was separated by 10% SDS-PAGE and analysed by Western blotting using antibodies against TDP1, TOP1 and actin. The bands were quantified using ImageJ software and relative TDP1 (B) and TOP1 (C) protein levels normalised to that obtained for RKO is shown ± STD for 3 independent experiments. mRNA (1 μg) purified from the CRC cell lines was reverse transcribed into cDNA that was used for real-time PCR analysis using primers against exon-crossing regions for TDP1 and TOP1. The relative quantity of mRNA (RQ value) for TDP1 (D) and TOP1 (E) was calculated as RQ = 2ΔΔCT and normalised to that obtained for the RKO cell line, the average from 3 independent experiments ± STD is depicted. A graphical representation of the Pearson’s correlation coefficient (R) between CRC TDP1 protein and mRNA levels is shown in (F) and for TOP1 protein and mRNA levels in (G).

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: SDS Page, Western Blot, Software, Purification, Reverse Transcription, Real-time Polymerase Chain Reaction

Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial.

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: Staining, Microarray

Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer. Sores were classified as none, low, moderate or high for TDP1 and TOP1 staining, with samples recorded if >5% cells stained accordingly.

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: Staining, Microarray

Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: RKO and SW480 cells transfected with non-targeting scrambled siRNA (Scram), a TDP1 siRNA pool (4 sequences) and/or a TOP1 siRNA pool (4 sequences) for 72 hr were subjected to a clonogenic survival assay during which they were continuously treated with CPT-11 for the duration of colony formation (7-12 days). The colonies were fixed, stained and counted and the surviving fraction calculated and depicted as an average of three independent experiments ± SEM for RKO (A) and SW480 (D). TDP1 siRNA treated RKO cells (B) and SW480 cells (E) were subjected to a survival assay for irradiation sensitivity and average surviving fraction of three independent experiments ± STD is shown. WCE (40 μg) generated from the siRNA treated RKO (C) and SW480 (F) cells was analysed further by 10% SDS-PAGE and Western blotting using antibodies against TDP1, TOP1 and actin. (G) Recombinant human TDP1 (2 pM) and WCE (25 ng, 30 ng and 35 ng) generated from either mock (Scram) or TDP1 siRNA treated RKO cells were subjected to an in vitro TDP1 activity assay and the reaction products separated on a 20% Urea Sequagel prior to imaging at 635 nm. The substrate (3′-PY) and product (3′-P) are indicated by arrows. The bands were quantified using Image J software and the average % cleavage for three independent experiments ± STD is shown in (H). TDP1 depletion was carried out in the RKO cell line using four separate TDP1 siRNA sequences (05 to 08). Cells were subsequently used in a clonogenic CPT-11 survival assay (I) or a gel-based TDP1 activity assay to measure knockdown efficiency (J). The average surviving fraction for two independent experiments ± STD is shown in (I). (K) TDP1 siRNA or mock (scram) treated RKO cells were treated with media containing either DMSO or 50 μM CPT-11 for 1 hr and then subjected to analysis by alkaline comet assay. The data shown is a representative single experiment ± STD for 100 cells per sample. (L) TDP1 siRNA or mock (scram) treated RKO cells on coverslips were treated with media containing DMSO or 1 μM CPT-11 for 1.5 hr and left to recover for 2 hr prior to fixing, permeabilising and staining with a γ-H2AX and a FITC labelled secondary antibody. γ-H2AX foci were visualised and analysed for 36 cells per sample on a Nikon Eclipse e-400 microscope. The average number of γ-H2AX foci per cell for three independent experiments is shown ± STD. Paired Student’s t-test (2-tailed) statistical analysis shown as *= p<0.05 and **= p<0.01.

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: Transfection, Clonogenic Cell Survival Assay, Staining, Irradiation, Generated, SDS Page, Western Blot, Recombinant, In Vitro, Activity Assay, Imaging, Software, Knockdown, Alkaline Single Cell Gel Electrophoresis, Microscopy

Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: CRC cell lines transfected with the indicated amounts of pCI-neo-myc vector or with pCI-neo-myc vector encoding human TDP1 were subjected to a clonogenic survival assay during which they were continuously treated with CPT-11 for the duration of colony formation (7-12 days). The colonies were fixed, stained and counted and the surviving fraction calculated and depicted as an average of three independent experiments ± STD for the CRC cell lines SW480 (A), RKO (C) and DLD1 (E). WCE (40 μg) generated from the SW480 (B), RKO (D) and DLD1 (F) cell lines overexpressing either pCI-neo-myc vector or that encoding human TDP1 was analysed by 10% SDS-PAGE and Western blotting using antibodies against TDP1, TOP1 and actin. The bands relating to overexpressed Myc-tagged TDP1 (TDP1-myc) and endogenous TDP1 (TDP1-end) are indicated. (G) TDP1 activity assay reactions containing Cy5.5 labelled substrate and WCE for SW480 (25 ng), RKO (10 ng) and DLD1 (25 ng) overexpressing indicated amounts of pCI-neo-myc vector or that encoding human TDP1 were separated on a 20% Urea Sequagel and imaged at 635 nm. A recombinant human TDP1 (4 pM) control was included. The substrate (3′-PY) and product (3′-P) are indicated by arrows. The bands were quantified using Image J software and the average % cleavage for three independent experiments ± STD is shown in (H). (I) RKO cells containing pCI-neo-myc empty vector or that encoding human TDP1 were treated with either DMSO or 50 μM CPT-11 for 1 hr prior to analysis by alkaline comet assay. Data shown is of three independent repeats ± STD. (J) RKO cells overexpressing pCI-neo-myc vector or that encoding human TDP1 plated on coverslips were treated with DMSO or 1 μM CPT-11 for 1.5 hr and left to recover for 2 hr prior to fixing, permeabilising and staining with a γ-H2AX and a FITC labelled secondary antibodies. γ-H2AX foci were visualised and analysed on a Nikon Eclipse e-400 microscope. The average number of γ-H2AX foci per cell for three independent experiments is shown ± STD. Paired Student’s t-test (2-tailed) statistical analysis shown as *= p<0.05 and **= p<0.01.

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: Transfection, Plasmid Preparation, Clonogenic Cell Survival Assay, Staining, Generated, SDS Page, Western Blot, Activity Assay, Recombinant, Control, Software, Alkaline Single Cell Gel Electrophoresis, Microscopy

Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: (A) Diagram depicting TDP1 processing of a 5′-Cy5.5 labelled oligonucleotide substrate harbouring a 3′-phosphotyrosine modification (3′-PY) to form a Cy5.5 labelled oligonucleotide product harbouring a 3′-phosphate (3′-P) that is smaller in size. (B) 10 uL reaction volumes containing Cy5.5 labelled substrate and the indicated amounts of recombinant human TDP1 (2 pM) or CRC WCE (ng) were separated on a 20% Urea Sequagel and imaged at 635 nm. The substrate (3′-PY) and product (3′-P) are indicated by arrows. (C) Reaction products were quantified using ImageJ software and the average % cleavage for three independent experiments ± STD is shown. A graphical representation of the Pearson’s correlation coefficient (R) between CRC TDP1 protein levels and % cleavage for 30 ng WCE (D) or 60 ng WCE (E).

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: Modification, Recombinant, Software

Journal: Molecular cancer therapeutics

Article Title: Clinical and cellular roles for TDP1 and TOP1 in modulating colorectal cancer response to irinotecan

doi: 10.1158/1535-7163.MCT-14-0762

Figure Lengend Snippet: (A) Adhered CRC cells seeded on 10 cm dishes were subjected to CPT-11 treatment for the duration of colony formation (7 to 12 days). Colonies were fixed, stained and counted and the surviving fraction calculated and depicted as an average of three independent experiments ± STD for each cell line. The Pearson’s correlation coefficient (R) between CRC surviving fraction at 0.5 μM (B) or 1.0 μM (C) CPT-11 treatment and TDP1 protein levels, TOP1 protein levels and TDP1/TOP1 protein level ratio is shown. For the latter, a correlation lacking data for SW48 cell lines (triangle) was also carried out (grey dotted line) (D) Irinotecan/radiotherapy response for rectal cancers obtained from the RICE trial was measured as non-responsive or responsive if pathologic complete remission (pCR) or residual tumour (micro-foci positive) was observed and the distribution of response for none/low TDP1 rectal cancers or moderate/high TDP1 rectal cancers is shown.

Article Snippet: We conclude from these observations that TDP1 and TOP1 expression also varies considerably in clinical CRC samples. fig ft0 fig mode=article f1 fig/graphic|fig/alternatives/graphic mode="anchored" m1 Open in a separate window Figure 2 caption a7 caption a8 TDP1 and TOP1 protein levels vary widely in CRC patient samples (A) A representative IHC image for TDP1 (NB100-81642, Novus Biologicals) in normal colon epithelium showing nuclear staining in crypt and in surface epithelium cells. (B) Representative IHC images for high and low TDP1 staining in CRC samples from a CRC Tissue Microarray (TMA). (C) Distribution of staining intensity for TDP1 and TOP1 (NCL-TOPO1, Leica Microsystems) IHC on CRC samples from a TMA. (D) Representative IHC images for high and no TDP1 staining for rectal cancers obtained from the RICE trial. (E) Distribution of staining intensity for TDP1 IHC on rectal cancer samples obtained from the RICE trial. table ft1 table-wrap mode="anchored" t5 caption a7 None Low Medium High Ascending 1 4 8 3 Caecum 0 2 1 0 Descendin 0 1 3 1 Rectum 0 3 2 2 Sigmoid 1 4 9 7 Transverse 0 2 4 1 None Low Medium High Ascending 0 4 8 4 Caecum 0 2 1 0 Descendin 0 1 2 1 Rectum 0 0 3 3 Sigmoid 0 4 7 10 Transverse 0 2 3 2 Open in a separate window caption a8 TDP1 and TOP1 staining in tumours selected from different colorectal anatomical sites Tissue microarray slides of paraffin embedded colorectal cancer samples with documented tumour demographic and correlated overall survival data were deparaffinised and stained with antibodies for TDP1 (NB100-81642, Novus Biologicals, CO, USA) and TOP1 (NCL-TOPO1, Leica Microsystems, Milton Keynes, UK), both at 1:200 dilution using a Bond Max Autostainer.

Techniques: Staining

Journal: Oncology Letters

Article Title: Chronic exposure to the gibberellin derivative GA-13315 sensitizes breast cancer MCF-7 cells but not colon cancer HCT116 cells to irinotecan

doi: 10.3892/ol.2020.12144

Figure Lengend Snippet: Sequences of oligos used for quantitative PCR.

Article Snippet: The membranes were incubated with primary antibodies against Top1 (1:10,000; cat. no. ab109374; Abcam), tyrosyl DNA phosphodiesterase 1 (Tdp1; 1:1,000; cat. no. 2360; Cell Signaling Technology, Inc.), Chk1 (cat. no. 59710; Cell Signaling Technology, Inc.), Bax (1:10,000; cat. no. ab32503; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam) and β-actin (1:5,000; cat. no. ab8227; Abcam) overnight at 4°C.

Techniques:

Journal: Oncology Letters

Article Title: Chronic exposure to the gibberellin derivative GA-13315 sensitizes breast cancer MCF-7 cells but not colon cancer HCT116 cells to irinotecan

doi: 10.3892/ol.2020.12144

Figure Lengend Snippet: Effect of 13-chlorine-3,15-dioxy-gibberellic acid methyl ester on the expression of proteins involved in the chemosensitivity of tumor cell lines. (A) Protein expression levels were normalized to β-actin and quantified using ImageJ v1.49 software, and (B) data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 weeks (control); # P<0.05 vs. 4 weeks; φ P<0.05 vs. 8 weeks. Top1, topoisomerase I; Tdp1, tyrosyl DNA phosphodiesterase 1; Chk1, checkpoint kinase 1; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were incubated with primary antibodies against Top1 (1:10,000; cat. no. ab109374; Abcam), tyrosyl DNA phosphodiesterase 1 (Tdp1; 1:1,000; cat. no. 2360; Cell Signaling Technology, Inc.), Chk1 (cat. no. 59710; Cell Signaling Technology, Inc.), Bax (1:10,000; cat. no. ab32503; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam) and β-actin (1:5,000; cat. no. ab8227; Abcam) overnight at 4°C.

Techniques: Expressing, Software, Standard Deviation, Control

Journal: Oncology Letters

Article Title: Chronic exposure to the gibberellin derivative GA-13315 sensitizes breast cancer MCF-7 cells but not colon cancer HCT116 cells to irinotecan

doi: 10.3892/ol.2020.12144

Figure Lengend Snippet: Effect of 13-chlorine-3,15-dioxy-gibberellic acid methyl ester on the mRNA expression levels of genes involved in the chemosensitivity of tumor cell lines. Reverse transcription-quantitative PCR analysis was performed to determine the mRNA expression levels of (A) Top1, (B) Chk1, (C) Bax and (D) Bcl-2 in MCF-7 and HCT116 cells. Data are presented as the mean ± standard deviation of three independent experiments. *P<0.05, **P<0.01 and ***P<0.001 vs. 0 week (control); # P<0.05 vs. 4 weeks; φ P<0.05 vs. 8 weeks. Top1, topoisomerase I; Chk1, checkpoint kinase 1; Bcl-2, B-cell lymphoma-2.

Article Snippet: The membranes were incubated with primary antibodies against Top1 (1:10,000; cat. no. ab109374; Abcam), tyrosyl DNA phosphodiesterase 1 (Tdp1; 1:1,000; cat. no. 2360; Cell Signaling Technology, Inc.), Chk1 (cat. no. 59710; Cell Signaling Technology, Inc.), Bax (1:10,000; cat. no. ab32503; Abcam), Bcl-2 (1:1,000; cat. no. ab32124; Abcam) and β-actin (1:5,000; cat. no. ab8227; Abcam) overnight at 4°C.

Techniques: Expressing, Reverse Transcription, Real-time Polymerase Chain Reaction, Standard Deviation, Control

Journal: Nucleic Acids Research

Article Title: TDP1 splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition

doi: 10.1093/nar/gkae1163

Figure Lengend Snippet: HAP1 cells are hypersensitive to camptothecin and deficient of TDP1 protein. ( A ) Cell viability dose-response curves of HAP1, human retinal pigment epithelial (RPE-1) and human osteosarcoma (U2OS) cell lines to camptothecin treatment. ( B ) Chemogenetic CRISPR-Cas9 knockout screens in RPE-1 , HEK293A and HAP1 cells subjected to camptothecin treatment, highlighting the relative impacts on gRNAs targeting TDP1 (•) and TDP2 (•) in the cell populations interpreted as Zscore or log2 fold-change. ( C, left ) Cell viability dose-response curves to camptothecin of HAP1 and ( D, left ) RPE-1 cells in the presence or absence of TDP2. For the comparison in (C, left ) at 2 nM camptothecin, the P-value (**** P < 0.0001) was calculated using two-way ANOVA. ( C & D, right ) Quantified area under curve (AUC) data from the corresponding dose-response curves. In ( A ), ( C ) & ( D ), 5000 HAP1 cells and 1000 RPE-1 and U2OS cells were seeded in technical duplicates 24 h before camptothecin treatment. Treated cells were incubated for 4 days before measuring the cell viability with Alamar Blue fluorescent indicator. Experimental data were the average of 3 biological repeats ± s.e.m. ( E ) Comparison of the differential AUCs (AUC TDP2-KO /AUC WT ) between ( C ) HAP1 and ( D ) RPE-1 cells. The P-value (*** P < 0.001) was calculated through an unpaired two-tailed t -test (i.e. one-way ANOVA). ( F ) Total cell lysates of individual cell lines were immunoblotted for TDP1 protein. PCNA serves as a loading control. The sizes (kDa) of reference proteins in PageRuler prestained protein ladder are indicated on the right. ( G ) Normalized read counts of TDP1 mRNA in HAP1, RPE-1 and U2OS cell lines. RNA read counts for each cell line were sourced from RNA-Seq data available online and analyzed using DESeq2. Each column represents an RNA-Seq experiment with multiple biological replicates (black dots, n = 2–4 ). Adjusted p-values of TDP1 RNA read count comparison against HAP1 cells are shown. ( H ) Comparison of TDP1 mRNA level (normalized against PUM1 as a stable housekeeping gene) between different cell lines via RT-qPCR. Normalized TDP1 mRNA levels were compared against HAP1 cells. Experimental data were the average of three technical replicates ± S.E.M.

Article Snippet: Antibodies used were: TDP1 (Bethyl Lab Inc., A301-618A; 1:1000), PCNA (Santa Cruz Biotechnology, sc56; 1:1000) and vinculin (Abcam, ab219649; 1:1000).

Techniques: CRISPR, Knock-Out, Comparison, Incubation, Two Tailed Test, Control, RNA Sequencing, Quantitative RT-PCR

Journal: Nucleic Acids Research

Article Title: TDP1 splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition

doi: 10.1093/nar/gkae1163

Figure Lengend Snippet: TDP1 splice variant impairs TDP1 RNA splicing and causes exon skipping in HAP1 cells. ( A ) Pairwise comparison of the sequenced non-coding DNA (intronic regions adjacent to the exon ends) of TDP1 in HAP1, U2OS and RPE-1 cells against the reference genome. Introns have been compressed into fixed lengths (50 bp width; black connecting lines) to showcase the entire TDP1 gene, where the blue horizontal rectangles are individual exons except for exon 6 (magenta rectangle). Genomic variants in the TDP1 non-coding sequences for individual cell lines are highlighted as a ‘lollipop’ and coloured according to the variant type: (•) variant in the intron , (•) variant that affects splice donor/acceptor site, (•) variant in the 5′ or 3′ untranslated region according to the whole-exome sequencing. ( B ) Sanger sequencing chromatograms of RPE-1, U2OS, HAP1 and HAP1-BE3 cells showing the genomic sequence of TDP1 around exon 6. The TDP1 c.660–1G > A splice-site mutation (‘AA’ splice acceptor site) is unique to HAP1 and HAP1-BE3 cells. ( C ) Sashimi plots of TDP1 RNA expression in HAP1 (top), U2OS (middle) and RPE-1 (bottom) cells where the peaks indicate the RNA coverage at the coding sequence of different exons and the peak height reflects the depth of mapped reads from RNA-Seq results. The peaks at exons 3, 6, 7 and 8 were annotated and the dotted line boxes highlight the peak heights of exons 6 and 7 in HAP1, U2OS and RPE-1 cell lines. ( D ) Top : Schematics of the expected PCR amplicon sizes (in bp) of multiple targeted PCRs – i, ii, iii and iv (with reverse primers annealing to exon 6, 7, 8 or 9 respectively) of the TDP1 cDNA, taking into account when exon 6 or exon 6 + 7 skipping occur. Note: forward primers annealing to exon 3 are not identical in different PCRs. Bottom : Separation of DNA products amplified using reverse transcribed TDP1 cDNA in different cell lines ( R : RPE-1, U : U2OS, H : HAP1, HB : HAP1-BE3) on a 2% agarose gel to test for TDP1 mRNA length and purity. The estimated size (bp) of DNA bands for each PCR (i, ii, iii and iv) is annotated (smaller PCR products due to exon skipping are annotated in magenta). The DNA ladder (bottom left) is made up of DNA bands at 100 base-pair intervals.

Article Snippet: Antibodies used were: TDP1 (Bethyl Lab Inc., A301-618A; 1:1000), PCNA (Santa Cruz Biotechnology, sc56; 1:1000) and vinculin (Abcam, ab219649; 1:1000).

Techniques: Variant Assay, Comparison, Sequencing, Mutagenesis, RNA Expression, RNA Sequencing, Amplification, Reverse Transcription, Agarose Gel Electrophoresis

Journal: Nucleic Acids Research

Article Title: TDP1 splice-site mutation causes HAP1 cell hypersensitivity to topoisomerase I inhibition

doi: 10.1093/nar/gkae1163

Figure Lengend Snippet: Correcting TDP1 splice-site mutation restores TDP1 protein expression and function in HAP1 cells. ( A ) Sanger sequencing chromatograms of two clones each from RPE-1, HAP1 and HAP1 STAR cells, where the HAP1 STAR clones had had their endogenous TDP1 splice acceptor mutant ‘AA’ successfully edited into functional ‘AG’ via DNA-templated HDR. Two clones (HAP1 STAR Clone #3 & #30) were identified. A silent mutation of TDP1 c.672C > T was also observed in the edited HAP1 STAR clones. This mutation was intentionally included in the editing template to minimize re-annealing of gRNA to the target DNA sequence upon successful genome editing. ( B ) Separation of DNA products amplified using reverse transcribed TDP1 cDNA in edited (HAP1 STAR) and unedited HAP1 clones as well as in RPE-1 clones on a 2% agarose gel to test for TDP1 mRNA length and purity. The estimated size (bp) of DNA bands without exon skipping (full length of mRNA) for each PCR (i, ii, iii and iv) is annotated. See top part of Figure . The presence of any DNA bands of smaller sizes demonstrate the phenomenon of exon skipping in TDP1 mRNA. The DNA ladder (left) is made up of DNA bands at 100 base-pair intervals. ( C ) Total cell lysates of HAP1 STAR and other unedited clones (HAP1 and RPE-1) were immunoblotted to detect the recovery of TDP1 protein expression upon editing. PCNA serves as a loading control. ( D ) Cell viability dose-response curves to camptothecin of HAP1 STAR clones relative to unedited HAP1 clones. Around 10 000 HAP1 cells were seeded in technical duplicates 24 h before camptothecin treatment. Treated cells were incubated for 3 days before measuring the cell viability with Alamar Blue fluorescent indicator. Experimental data were the average of three biological repeats ± S.E.M. For the comparison against ‘WT’ HAP1 at 10 nM camptothecin, the P-value (**** P < 0.0001) was calculated using two-way ANOVA. ( E ) Quantified AUC based on the cell viability dose-response curves in D. The P-value (**** P < 0.0001) was calculated through ordinary one-way analysis of variance (ANOVA). Despite showing only the comparison of HAP1 STAR clones #3 and #30 against the unedited HAP1 polyclonal cells, their comparison against unedited HAP1 clones (#3 and #5) were also significant with P-value ≤ 0.0001. ( F ) Representative images of alkaline ‘comets’ for ‘WT’ HAP1, HAP1 TDP1 KO , HAP1 STAR #3, and HAP1 STAR #30 upon different treatment conditions (UNT: untreated, CPT: camptothecin, H 2 O 2 : hydrogen peroxide). H 2 O 2 (100 μM) treatment was included as a positive control for DNA single-stranded break induction. ( G ) Quantified Olive tail moment of the ‘comets’, which reflects the amount of DNA single-stranded breaks. Experimental data were the mean (bar height) of the population medians in 3 biological repeats (dots) ± S.E.M. 82–150 comets were analyzed for each condition in each repeat. Different cell lines were compared against ‘WT’ HAP1 in each treatment condition using ordinary two-way ANOVA with the following reported P-values : (ns) P > 0.05 and (****) P < 0.0001.

Article Snippet: Antibodies used were: TDP1 (Bethyl Lab Inc., A301-618A; 1:1000), PCNA (Santa Cruz Biotechnology, sc56; 1:1000) and vinculin (Abcam, ab219649; 1:1000).

Techniques: Mutagenesis, Expressing, Sequencing, Clone Assay, Functional Assay, Amplification, Reverse Transcription, Agarose Gel Electrophoresis, Control, Incubation, Comparison, Positive Control

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: TDP1 and TOP1 as targets in anticancer treatment of NSCLC: Activity and protein level in normal and tumor tissue from 150 NSCLC patients correlated to clinical data.

doi: 10.1016/j.lungcan.2021.12.010

Figure Lengend Snippet: Fig. 1. Overview of study design and exclusion of patients. A) Graphical pre sentation of experimental setup. Fresh frozen tissue and formalin fixed and paraffin embedded (FFPE) tissue were used for the experiments. For the fresh frozen biopsies, the first and the last section were HE stained and used for microscopic evaluation of the morphology of the normal and tumor tissue. The sections between the HE stained sections were transferred to an Eppendorf tube for tissue extraction. The tissue extract was then used for measurement of TDP1 and TOP1 activity with biosensors and protein con centration with ELISA. FFPE tumor tissue was used for TDP1 and TOP1 immunohisto chemistry to evaluate staining intensity in normal and tumor cells. Created with Bio Render.com. B) Exclusion of patient samples. Patient samples were excluded based on abnormal morphology or technical issues. The sample number (n) represents the num ber of patient samples.

Article Snippet: TDP1 and TOP1 protein concentration were measured using sandwich ELISA kits from CUSABIO and Cloud-Clone Corp., respectively.

Techniques: Staining, Extraction, Activity Assay, Enzyme-linked Immunosorbent Assay, Immunohistochemistry

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: TDP1 and TOP1 as targets in anticancer treatment of NSCLC: Activity and protein level in normal and tumor tissue from 150 NSCLC patients correlated to clinical data.

doi: 10.1016/j.lungcan.2021.12.010

Figure Lengend Snippet: Fig. 2. TDP1 and TOP1 activity in normal and tumor tissue from 122 NSCLC patients. A) Comparison of TDP1 activity in normal and tumor tissue. The line represents the median of the activity measurements. B) Comparison of TOP1 activity in normal and tumor tissue. The line represents the median of the activity measurements. C) Correlation of TDP1 and TOP1 activity in the normal samples. D) Correlation of TDP1 and TOP1 activity in the tumor samples. E) Correlation of the tumor/normal activity ratio for TDP1 and TOP1. The tumor/normal activity ratio was calculated as activity in tumor samples divided by activity in normal samples (T/N). T = tumor tissue, N = normal tissue.

Article Snippet: TDP1 and TOP1 protein concentration were measured using sandwich ELISA kits from CUSABIO and Cloud-Clone Corp., respectively.

Techniques: Activity Assay, Comparison

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: TDP1 and TOP1 as targets in anticancer treatment of NSCLC: Activity and protein level in normal and tumor tissue from 150 NSCLC patients correlated to clinical data.

doi: 10.1016/j.lungcan.2021.12.010

Figure Lengend Snippet: Fig. 3. TDP1 and TOP1 protein concentration in normal and tumor tissue from 122 NSCLC patients. A) Comparison of TDP1 protein concentrations in normal and tumor tissue. The line represents the median of the protein concentrations measurements. B) Comparison of TOP1 protein concentrations in normal and tumor tissue. The line represents the median of the protein concentrations measurements. C) Correlation of TDP1 and TOP1 protein concentrations in the normal samples. D) Correlation of TDP1 and TOP1 protein concentrations in the tumor samples. E) Correlation of the tumor/normal protein ratio for TDP1 and TOP1. The tumor/ normal protein ratio was calculated as protein concentration in tumor samples divided by protein concentration in normal samples (T/N). T = tumor tissue, N = normal tissue.

Article Snippet: TDP1 and TOP1 protein concentration were measured using sandwich ELISA kits from CUSABIO and Cloud-Clone Corp., respectively.

Techniques: Protein Concentration, Comparison

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: TDP1 and TOP1 as targets in anticancer treatment of NSCLC: Activity and protein level in normal and tumor tissue from 150 NSCLC patients correlated to clinical data.

doi: 10.1016/j.lungcan.2021.12.010

Figure Lengend Snippet: Fig. 4. Correlation of TDP1 and TOP1 activity and protein concentrations from normal and tumor tissue from 122 NSCLC patients. A) Correlation of TDP1 activity and protein concentration in normal tissue. B) Correlation of TOP1 activity and protein concentration in normal tissue. C) Correlation of TDP1 activity and protein concentration in tumor tissue. D) Correlation of TOP1 activity and protein concentration in tumor tissue. E) Correlation of the tumor/normal activity and protein ratio for TDP1. F) Correlation of the tumor/normal activity and protein ratio for TOP1. For E) and F) the tumor/normal activity and protein ratios were calculated as activity or protein concentration in tumor samples divided by activity or protein concentration in normal samples (T/N). T = tumor tissue, N = normal tissue.

Article Snippet: TDP1 and TOP1 protein concentration were measured using sandwich ELISA kits from CUSABIO and Cloud-Clone Corp., respectively.

Techniques: Activity Assay, Protein Concentration

Journal: Lung cancer (Amsterdam, Netherlands)

Article Title: TDP1 and TOP1 as targets in anticancer treatment of NSCLC: Activity and protein level in normal and tumor tissue from 150 NSCLC patients correlated to clinical data.

doi: 10.1016/j.lungcan.2021.12.010

Figure Lengend Snippet: Fig. 5. TDP1 and TOP1 immunohistochemistry on FFPE tumor tissue from NSCLC patients. A) Representative pictures of TDP1 and TOP1 immunohisto chemistry with staining intensity in tumor (T) cells compared to normal (N) cells. The arrows point to pictures focusing on normal cells or tumor cells. B) Repre sentative pictures of NSCLC tumor tissue with immune cells staining for TDP1 and TOP1. The arrows point to pictures focusing on the immune cells.

Article Snippet: TDP1 and TOP1 protein concentration were measured using sandwich ELISA kits from CUSABIO and Cloud-Clone Corp., respectively.

Techniques: Immunohistochemistry, Staining

Journal: Cell Death & Disease

Article Title: H263A and SCAN1/H493R mutant TDP1 block TOP1-induced double-strand break repair during gene transcription in quiescent cells and promote cell death

doi: 10.1038/s41419-025-08085-y

Figure Lengend Snippet: a Protein blot of TDP1 in serum-starved TDP1 +/+ and TDP1 −/− RPE-1 cells complemented with an empty vector (EV), wild-type TDP1 (WT), TDP1 H263A (H263A) or TDP1 H493R (H493R) is shown. Quantification represented as mean ± SEM ( n = 3 independent experiments). nd not detectable. b 53BP1 foci in serum-starved complemented cells treated with CPT (12.5 μM) for 1 h. Where indicated, cells were pre-incubated with DRB (100 μM) for 3 h prior to CPT treatment. Representative images of 53BP1 (red) and γH2AX (green) foci and DAPI counterstain (blue) are shown. c , d 53BP1 foci in serum-starved complemented cells after 1 h treatment with 12.5 μM CPT, and during repair in drug-free medium. Representative images for the 24 h of repair time point ( c ) and a protein blot of TDP1 ( d ) are shown. A low doxycycline dose was used in d . Other details as in a and b . e Detection of DSBs by neutral comet assay in serum-starved complemented cells treated with CPT (25 μM) for 1 h and after 24 h repair in drug-free medium. Representative images of nuclei are shown. f Chromosomal breaks were quantified in serum-starved complemented cells treated with CPT (25 μM) for 2 h followed by 24 h repair in drug-free medium and fused with HeLa cells synchronized in metaphase. A representative image of chromosomal breaks is shown. Black arrows indicate chromosomal breaks. UNT untreated. n = 3 independent experiments. Data were represented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t -test for b and f , two-way ANOVA followed by Tukey’s multiple comparisons test for c and d , and ratio paired t -test for e . ns non-significance. Scale bar, 10 μm for b , c and f and 50 μm for e .

Article Snippet: TDP1 (Proteintech, 10641-1-AP) and TOP1cc (Millipore, MABE1084) antibodies were used.

Techniques: Plasmid Preparation, Incubation, Neutral Comet Assay, Two Tailed Test

Journal: Cell Death & Disease

Article Title: H263A and SCAN1/H493R mutant TDP1 block TOP1-induced double-strand break repair during gene transcription in quiescent cells and promote cell death

doi: 10.1038/s41419-025-08085-y

Figure Lengend Snippet: Analysis of TOP1 cleavage-complexes (TOP1cc) ( a , c , e , g , i , and j ) and FLAG (TDP1) ( b , d , f , and h ) by ICE assay. Serum-starved TDP1 −/− RPE-1 cells complemented with an empty vector (EV), wild-type TDP1 (WT), TDP1 H263A (H263A) or TDP1 H493R (H493R) were treated with CPT (25 μM) for 1 h followed by repair in drug-free medium where indicated. Where indicated, cells were pre-incubated with DRB (100 μM) for 3 h prior to CPT treatment. Top , quantification. Bottom , representative plots of TOP1cc and FLAG are shown. UNT untreated. n = 3 independent experiments. Data were represented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t -test. ns non-significance.

Article Snippet: TDP1 (Proteintech, 10641-1-AP) and TOP1cc (Millipore, MABE1084) antibodies were used.

Techniques: Plasmid Preparation, Incubation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: H263A and SCAN1/H493R mutant TDP1 block TOP1-induced double-strand break repair during gene transcription in quiescent cells and promote cell death

doi: 10.1038/s41419-025-08085-y

Figure Lengend Snippet: a Protein blot of TDP1 in serum-starved TDP1 +/+ RPE-1 cells complemented with an empty vector (EV), wild-type TDP1 (WT), TDP1 H263A (H263A) or TDP1 H493R (H493R) is shown. b 53BP1 foci in serum-starved complemented cells treated with CPT (12.5 μM) for 1 h. c 53BP1 foci in serum-starved complemented cells after 1 h treatment with 12.5 μM CPT, and during repair in drug-free medium. d , e Analysis of TOP1 cleavage-complexes (TOP1cc) ( d ) and FLAG (TDP1) ( e ) by ICE assay. Serum-starved complemented cells were treated with CPT (25 μM) for 1 h. Top , quantification. Bottom , representative plots of TOP1cc and FLAG are shown. UNT untreated. n = 3 independent experiments. Data were represented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t -test for ( b ), ( d ) and ( e ), and two-way ANOVA followed by Tukey’s multiple comparisons test for ( c ). ns non-significance.

Article Snippet: TDP1 (Proteintech, 10641-1-AP) and TOP1cc (Millipore, MABE1084) antibodies were used.

Techniques: Plasmid Preparation, Two Tailed Test

Journal: Cell Death & Disease

Article Title: H263A and SCAN1/H493R mutant TDP1 block TOP1-induced double-strand break repair during gene transcription in quiescent cells and promote cell death

doi: 10.1038/s41419-025-08085-y

Figure Lengend Snippet: TDP1 +/+ and TDP1 −/− mock-depleted (sg AAVS1 ) or TDP2-depleted (sg TDP2 ) RPE-1 cells. a Protein blot of TDP1 and TDP2 is shown. b 53BP1 foci in serum-starved cells treated with CPT (12.5 μM) for 1 h. c 53BP1 foci in serum-starved cells after 1 h treatment with 12.5 μM CPT, and during repair in drug-free medium. d Analysis of TOP1 cleavage-complexes (TOP1cc) by ICE assay. Serum-starved cells were treated with CPT (25 μM) for 1 h followed by 1 h repair in drug-free medium. Top , quantification. Bottom , representative plot of TOP1cc is shown. UNT untreated. n = 3 independent experiments. Data were represented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t -test for ( b ) and ( d ), and two-way ANOVA followed by Tukey’s multiple comparisons test for ( c ).

Article Snippet: TDP1 (Proteintech, 10641-1-AP) and TOP1cc (Millipore, MABE1084) antibodies were used.

Techniques: Two Tailed Test

Journal: Cell Death & Disease

Article Title: H263A and SCAN1/H493R mutant TDP1 block TOP1-induced double-strand break repair during gene transcription in quiescent cells and promote cell death

doi: 10.1038/s41419-025-08085-y

Figure Lengend Snippet: Model for TOP1-induced DSB repair and TDP1 H493R and TDP1 H263A -associated genome instability and reduced survival.

Article Snippet: TDP1 (Proteintech, 10641-1-AP) and TOP1cc (Millipore, MABE1084) antibodies were used.

Techniques:

Journal: Cell Death & Disease

Article Title: H263A and SCAN1/H493R mutant TDP1 block TOP1-induced double-strand break repair during gene transcription in quiescent cells and promote cell death

doi: 10.1038/s41419-025-08085-y

Figure Lengend Snippet: a Translocation frequencies were quantified in serum-starved TDP1 +/+ and TDP1 −/− mock-depleted (sg AAVS1 ) or TDP2-depleted (sg TDP2 ) RPE-1 cells in metaphase spreads prepared 48 h after CPT treatment (25 μM) for 2 h followed by 6 h repair in drug-free medium. Workflow and a representative image of a chromosomal translocation are shown. White arrow indicates a translocation event. b Translocation frequencies were quantified in serum-starved TDP1 -/- RPE-1 cells complemented with an empty vector (EV), wild-type TDP1 (WT), TDP1 H263A (H263A) or TDP1 H493R (H493R). Other details as in ( a ). c – e Clonogenic survival of serum-starved TDP1 +/+ and TDP1 −/− mock-depleted (sg AAVS1 ) or TDP2-depleted (sg TDP2 ) cells ( c ), or TDP1 +/+ and TDP1 −/− RPE-1 cells complemented with an empty vector (EV), wild-type TDP1 (WT), TDP1 H263A (H263A) or TDP1 H493R (H493R) ( d , e ), treated with CPT (6.25 μM for c and e ) for 2 h, and after 6 h repair in drug-free media. A low doxycycline dose was used in e . Where indicated, cells were pre-incubated with DRB (100 μM) for 3 h prior to CPT treatment. After repair, cells were collected and re-cultured in serum-containing media. Workflow is shown in c . n = 3 independent experiments. Data were represented as mean ± SEM. Statistical significance was determined by two-tailed unpaired t -test for ( a–c and e ) and by two-way ANOVA followed by Tukey’s multiple comparisons test for ( d ). ns non-significance. Scale bar, 10 μm.

Article Snippet: TDP1 (Proteintech, 10641-1-AP) and TOP1cc (Millipore, MABE1084) antibodies were used.

Techniques: Translocation Assay, Plasmid Preparation, Incubation, Cell Culture, Two Tailed Test