Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1 interacts with KU70 and KU80 proteins. ( A ) Purification of BCAT1 interacting partners. Expression of BCAT1 and tubulin was analyzed by immunoblotting (top) in CUTLL1 T-ALL cells stably expressing empty vector or double epitope-tagged BCAT1 (myc/DDK). Cytoplasmic extracts prepared from BCAT1 myc/DDK-expressing or mock-transduced (CTRL) CUTLL1 cells were subjected to sequential immunoprecipitation (IP) using anti-FLAG and anti-MYC beads. Proteins were resolved by SDS-PAGE and visualized by silver staining (bottom). Molecular weights (Mr) are indicated on the left. ( B ) Top proteins interacting with BCAT1 identified by mass spectrometry based on the number of unique peptides and intensity (bubble plot). These proteins were not identified in the control IP. Results are from one experiment of two performed with similar results. ( C ) ShinyGO 0.8 software was used for enrichment analysis of BCAT1-interacting partners. KEGG pathway (top) and hallmark (bottom) analysis representing top significantly enriched pathways (lollipop plots). ( D ) Cytoplasmic extracts prepared from CUTLL1 T-ALL cells stably expressing empty vector or BCAT1 myc/DDK were subjected to IP using anti-FLAG beads. FLAG peptide was used to elute proteins from the beads and were resolved by SDS-PAGE. Immunoblotting for BCAT1, KU70, and KU80 proteins was performed. ( E ) HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with GFP-KU70 (left) or GFP-KU80 (right) expression vectors, and lysates were subjected to co-immunoprecipitation (Co-IP) using anti-MYC tag beads. Immunoblot analysis for BCAT1 (anti-BCAT1) and KU70 or KU80 (anti-GFP) was performed. Asterisk (*) indicates non-specific bands. ( F ) HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with GFP-KU70 (left) or GFP-KU80 (right) expression vectors, and lysates were subjected to Co-IP using anti-GFP beads. Immunoblot analysis for BCAT1 (anti-BCAT1 and anti-Flag) and KU70 or KU80 (anti-GFP) was performed. ( G ) Interaction between endogenously expressed BCAT1, KU70, and KU80 proteins in CCRF-CEM cells was demonstrated by IP with IgG (mIgG) and anti-BCAT1 antibody followed by immunoblot analysis with the indicated antibodies. DNA-PKcs was also detected. ( H ) Cellular localization analysis of BCAT1, KU70, and KU80 via Western blot analysis of nuclear and cytoplasmic cell fractions in cell lysates from CCRF-CEM T-ALL cells. Tubulin and Max proteins are shown as controls for cytosolic and nuclear fractions. CYT: cytoplasmic fraction; NUC: nuclear fraction. ( I ) Localization of BCAT1 (red) and KU70 (green) by immunofluorescence in CCRF-CEM and Jurkat T-ALL cells. DAPI (blue) was used as a nuclear marker. A scale bar is shown. ( J ) The interaction between BCAT1 and KU70 was assessed under basal conditions (top) and after 24 h of treatment with etoposide (1 µM; bottom) by proximity ligation assay (PLA) with the indicated pairs of primary antibodies. DAPI (blue) was used as a nuclear marker. Scale bar is shown.

Article Snippet: Cells were blocked in PBS-1% goat serum (S-1000-20; Vector Laboratories, Newark, NJ, USA) and incubated with rabbit antibodies against KU70 (10723-1-AP; 1:80; Proteintech Europe) and a mouse antibody against BCAT1 (1:80; BD Pharmingen) overnight at 4 °C.

Techniques: Purification, Expressing, Western Blot, Stable Transfection, Plasmid Preparation, Immunoprecipitation, SDS Page, Silver Staining, Mass Spectrometry, Control, Software, Transfection, Co-Immunoprecipitation Assay, Immunofluorescence, Marker, Proximity Ligation Assay

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1–depletion induces a dysfunctional DNA damage response following etoposide treatment. ( A ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of XRCC6 (top). vWA: von Willebrand A domain; SAP: SAF-A/B, Acinus, and PIAS domain. HEK 293T cells stably expressing epitope-tagged BCAT1 were transfected with the indicated plasmid. Cell lysates were subjected to IP with anti-FLAG beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non–specific bands. ( B ) Schematic representations of the plasmids encoding full-length (WT) and truncation mutants of BCAT1 (top). N: Branched–chain amino acid aminotransferase-like N-terminal domain; AT–IV: aminotransferase class IV domain; C: Branched-chain amino acid aminotransferase-like C–terminal domain. HEK 293T cells were transfected with HA–tagged XRCC6 and the indicated BCAT1 mutant plasmids. Cell lysates were subjected to IP with anti-HA beads followed by immunoblot analysis with the indicated antibodies. The arrows indicate expected positions of the respective proteins, and asterisks (*) indicate non-specific bands. ( C – E ) CCRF–CEM T-ALL cells transduced with shCTRL or sh BCAT1 were treated with 1 µM etoposide for the indicated time. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for proteins implicated in ( C , D ) the activation of the DNA damage response (pDNA-PKcs, pATM, pCHK1, pCHK2, pTP53); ( E ) DNA damage (γH2AX) and apoptosis (cleaved PARP-1). Total DNA–PKcs and ATM are shown as loading controls ( C ). Total CHK2, total TP53, and GADPH are shown as loading controls ( D , E ). Phospho-protein/protein ratios are shown (top) in each panel. A graphical representation of the phospho-protein/protein ratios is also shown for selected proteins (right panels).

Article Snippet: Cells were blocked in PBS-1% goat serum (S-1000-20; Vector Laboratories, Newark, NJ, USA) and incubated with rabbit antibodies against KU70 (10723-1-AP; 1:80; Proteintech Europe) and a mouse antibody against BCAT1 (1:80; BD Pharmingen) overnight at 4 °C.

Techniques: Stable Transfection, Expressing, Transfection, Plasmid Preparation, Western Blot, Mutagenesis, Transduction, Activation Assay

Journal: International Journal of Molecular Sciences

Article Title: BCAT1 Associates with DNA Repair Proteins KU70 and KU80 and Contributes to Regulate DNA Repair in T-Cell Acute Lymphoblastic Leukemia (T-ALL)

doi: 10.3390/ijms252413571

Figure Lengend Snippet: BCAT1-depletion decreases DNA repair by modulating KU70 acetylation levels. ( A ) Jurkat reporter cell lines were generated from parental cell lines by transfection of the pimEJ5-GFP construct and subsequent selection with puromycin for over 14 days. These cell lines were subsequently engineered to lose BCAT1 expression (pLKO.1 sh BCAT1 #3). The reporter cell lines were then electroporated with the pCBA-SceI endonuclease-expressing vector (or empty vector). After 72 h, the activity of the c-NHEJ (pimEJ5-GFP vector-expressing cells) DNA repair pathway was assessed by measuring the percentage of GFP-positive cells using flow cytometry. Error bars indicate ±SD. Results from one of three independent experiments performed with 6–9 replicates are shown. Significance was calculated using an unpaired Mann–Whitney U test. * p < 0.05. ( B ) U2OS cells were engineered to overexpress BCAT1 (BCAT1 myc/DDK). Cells were then transfected with the pimEJ5-GFP vector and pCBA-SceI or empty vector. After 48 h, the activity of the c-NHEJ (pimEJ5–GFP vector-expressing cells) DNA repair pathway was assessed by measuring the percentage of GFP-positive cells using flow cytometry. Error bars indicate ± SD. Results from one of two independent experiments are shown. Significance was calculated using an unpaired Mann-Whitney U test. *** p < 0.001. ( C ) U2OS cells were engineered to overexpress BCAT1 (BCAT1 myc/DDK) or BCAT1 mutants (K222A, SXXS). Cells were then transfected with the pimEJ5-GFP vector and pCBA-SceI or empty vector. After 48 h, the activity of the c-NHEJ (pimEJ5–GFP vector-expressing cells) DNA repair pathway was assessed by measuring the percentage of GFP–positive cells using flow cytometry. Error bars indicate ± SD. Results from one of two independent experiments are shown. Significance was calculated using an unpaired Mann-Whitney U test. * p < 0.05, ** p < 0.01. ( D ) Kinetics of DNA repair in CCRF-CEM control and BCAT1 stable knockdown T-ALL cells (sh BCAT1 #1, sh BCAT1 #2). The number of γH2AX foci (left), 53BP1 foci (middle), and coincident γH2AX/53BP1 foci (right) per nucleus following etoposide treatment are denoted. Each point represents data from a single cell, and the bars denote the median foci number per cell. Top panels: Significance was calculated using the Kruskal-Wallis test. ** p < 0.01, *** p < 0.001. n.s. = not significant. Box–and–whisker plots denote expression from minimum to maximum (bottom). Significance was calculated using an unpaired Mann-Whitney U test. ** p < 0.01, *** p < 0.001. n.s. = not significant. ( E ) CCRF-CEM T-ALL cells (left) were treated with different doses of ERG245 (100–200 µM) for 24 h. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for the indicated proteins. Total KU70 and GADPH are shown as loading controls. Jurkat T-ALL cells (right) were treated with different doses of ERG245 (100–300 µM) or Trichostatin A (TSA; 100 nM) for 24 h. Subsequently, whole cell lysates were collected and analyzed by immunoblotting for the indicated proteins. Total KU70 and GADPH are shown as loading controls. The acetylated KU70/total KU70 protein ratios and γH2AX/GADPH protein ratios are also shown. ( F ) Whole cell lysates from ΔE-NOTCH1 leukemias wild-type and KO for Bcat1 were immunoprecipitated using anti-acetyl-lysine affinity beads or control beads and probed for Ku70 and Bcat1. α-Tubulin is shown as a loading control (input).

Article Snippet: Cells were blocked in PBS-1% goat serum (S-1000-20; Vector Laboratories, Newark, NJ, USA) and incubated with rabbit antibodies against KU70 (10723-1-AP; 1:80; Proteintech Europe) and a mouse antibody against BCAT1 (1:80; BD Pharmingen) overnight at 4 °C.

Techniques: Generated, Transfection, Construct, Selection, Expressing, Plasmid Preparation, Activity Assay, Flow Cytometry, MANN-WHITNEY, Control, Knockdown, Whisker Assay, Western Blot, Immunoprecipitation

Journal: Clinical and Translational Science

Article Title: Screening and characterization of myositis‐related autoantibodies in COVID ‐19 patients

doi: 10.1111/cts.13434

Figure Lengend Snippet: Immunoprecipitation followed by Western blot was performed to visualize specific proteins recognized by autoantibodies from patient sera. (a) Immunoprecipitation of Ku70 protein. (b) Immunoprecipitation of Ku80 protein. (c) Immunoprecipitation of PL‐7 protein. Molecular weight is indicated on the left‐hand side of each figure.

Article Snippet: Ku‐70 polyclonal antibody (10723‐1‐AP), Ku‐80 polyclonal antibody (16389‐1‐AP), and TARS polyclonal antibody (14773‐1‐AP) were purchased from the Proteintech Group, Inc.

Techniques: Immunoprecipitation, Western Blot, Molecular Weight

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A Schematic diagram of pre-treatment tissue proteome sequencing for CRC patients receiving neoadjuvant therapy. B Volcano diagram of differential proteins between CR group and SD group patients. C Volcano diagram of differential proteins between PR group and SD group patients. D The Venn diagram shows the common differential proteins between the CR/SD group and the PR/SD group. E Waterfall plot shows the changes in tumor burden of CRC patients undergoing radiotherapy from initiation to the best objective response. Patients were grouped based on the expression of CNOT7 in tumor tissue. Scale bar, 120 μm. F Table and histogram recapitulating the outcomes of 45 CRC patients who received radiotherapy. G Representative magnetic resonance (MR) images of the rectum showing tumor changes at baseline and the best response after radiotherapy. Red arrows indicate the lesions. The panels display the CNOT7 status and therapeutic evaluation. H Representative immunohistochemical images of CNOT7. Right scale bar, 100 μm. Left scale bar, 30 μm. I Western blotting of CNOT7 in CRC and adjacent normal tissues. J CNOT7 mRNA levels in CRC and adjacent normal tissues. K , L Kaplan-Meier curves were generated on the www.kmplot.com website to show the post-progression survival ( K ) and overall survival ( L ) in III and IV stage CRC patients with low or high CNOT7 expression.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: Sequencing, Expressing, Immunohistochemical staining, Western Blot, Generated

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A – D CCK8 assay revealed that CNOT7 knockdown increased the radiation therapy sensitivity of CRC cells and CNOT7 overexpression promoted CRC cell resistance to radiation therapy. E , F Representative images and corresponding survival fraction curves of colony formation assays showed that CNOT7 knockdown increased radiotherapy sensitivity in HCT116. G , H Representative images and corresponding survival fraction curves of colony formation assays showed that overexpression of CNOT7 promotes radiation resistance in SW480. I , J Flow cytometry was employed to access the proportion of apoptosis in HCT116 and CNOT7 knockdown HCT116 cells with or without radiotherapy. K Flow cytometry was used to evaluate the proportion of apoptosis in SW480 and CNOT7 knockdown SW480 cells with or without radiotherapy. L Gross images of HCT116 and CNOT7 knockdown HCT116 cell-derived subcutaneous tumors with or without radiotherapy. M – O Tumor growth curves ( M ), tumor volume ( N ) and tumor weights ( O ) of HCT116 and CNOT7 knockdown HCT116 cell-derived subcutaneous tumors with or without radiotherapy.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: CCK-8 Assay, Knockdown, Over Expression, Flow Cytometry, Derivative Assay

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A The volcano plot shows the differential gene expression between HCT116 and CNOT7 knockdown HCT116 cells in RNA-seq analysis. B GO analysis was performed on differential genes. C GSEA analysis of DNA damage repair-related pathways. D , E Representative images and quantitation of γH2AX positive nuclei in HCT116 and CNOT7 knockdown HCT116 cells at different time points. F , G Representative images and quantitation of γ-H2AX positive nuclei in SW480 and CNOT7 overexpression SW480 cells at different time points. H Western blotting of CNOT7 and γ-H2AX protein expression at different time points after radiation treatment in HCT116 and CNOT7 knockdown HCT116 cell lines. I Western blotting of CNOT7 and γ-H2AX protein expression at different time points after radiation treatment in SW480 and CNOT7 overexpression SW480 cell lines. J , K Representative images of comet assay and quantitative analysis of tail moment in HCT116 and CNOT7 knockdown HCT116 cells at different time points. L , M Representative images of comet assay and quantitative analysis of tail moment in SW480 and CNOT7 overexpression SW480 cells at different time points.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: Gene Expression, Knockdown, RNA Sequencing, Quantitation Assay, Over Expression, Western Blot, Expressing, Single Cell Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A Western blotting of HR and NHEJ related protein expression at different time points after radiation treatment in HCT116 and CNOT7 knockdown HCT116 cell lines. B Western blotting of homologous recombination (HR) and non-homologous end joining (NHEJ) related protein expression at different time points after radiation treatment in SW480 and CNOT7 overexpression SW480 cell lines. C RT-qPCR analysis of the expression levels of homologous recombination (HR) and non-homologous end joining (NHEJ) related genes in HCT116 and CNOT7 knockdown HCT116 cell lines. D RT-qPCR analysis of the expression levels of homologous recombination (HR) and non-homologous end joining (NHEJ) related genes in SW480 and CNOT7 overexpression SW480 cell lines. E , F NHEJ and HR repair efficiency were assessed in HCT116 cells transfected with DR-GFP and EJ5-GFP reporter plasmids. G Representative immunohistochemistry (IHC) images showing the expression of CNOT7, XRCC6, and 53BP1 in HCT116 and CNOT7 knockdown HCT116 cell-derived subcutaneous tumors with or without radiotherapy. Scale bar: 60 μm.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: Western Blot, Expressing, Knockdown, Homologous Recombination, Non-Homologous End Joining, Over Expression, Quantitative RT-PCR, Transfection, Immunohistochemistry, Derivative Assay

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A Prediction of the interaction between XRCC6 and CNOT7 using AlphaFold 3. B , C Co-immunoprecipitation (Co-IP) analysis of CNOT7-XRCC6 interaction in HCT116 and SW480 cells. Lysates immunoprecipitated with IgG and anti-CNOT7 antibody. Immunoblots probed with Anti-XRCC6 and Anti-CNOT7 antibodies. D , E Co-IP analysis of CNOT7-XRCC6 interaction in HCT116 and SW480 cells. Lysates immunoprecipitated with IgG and anti-XRCC6 antibody. Immunoblots probed with Anti-XRCC6 and Anti-CNOT7 antibodies. F Detection of XRCC6 protein and RNA levels under CNOT7 overexpression in 293 T cells. G Western blotting of XRCC6 expression in HCT116 and CNOT7 knockdown HCT116 cells treated with cycloheximide (CHX 100 µg/ml). H Western blotting of XRCC6 expression in SW480 and CNOT7 knockdown SW480 cells treated with cycloheximide (CHX 100 µg/ml). I Western blotting of XRCC6 in 293 T and CNOT7 overexpression 293 T cells treated with cycloheximide (CHX 100 µg/ml). J Western blotting of XRCC6 and CNOT7 expression in HCT116 and CNOT7 knockdown HCT116 cells treated with MG132 (10 µM) or CQ (50 µM). K Western blotting of XRCC6 and CNOT7 in 293 T cells treated with MG132 (10 µM). L Western blotting of XRCC6 and CNOT7 expression in 293 T cells treated with CQ (50 µM). M Ubiquitination assay of XRCC6 performed in HCT116 and CNOT7 knockdown HCT116 cells. N Ubiquitination assay of XRCC6 performed in SW480 and CNOT7 knockdown SW480 cells. O Ubiquitination assay of XRCC6 performed in HCT116 and CNOT7 overexpression HCT116 cells. P Ubiquitination assay of XRCC6 performed in 293 T cells.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Over Expression, Expressing, Knockdown, Ubiquitin Proteomics

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A Immunoprecipitation-Mass Spectrometry (IP-MS) of IgG and CNOT7 antibody in HCT116. B The interacting proteins identified by IP-MS analysis. C Prediction of the interaction between XRCC6 and TRIM21 using AlphaFold 3. D , E Co-IP analysis of TRIM21-XRCC6 interaction in HCT116 and SW480 cells. Lysates immunoprecipitated with IgG and anti-TRIM21 antibody. Immunoblots probed with Anti-XRCC6 and Anti-TRIM21 antibodies. F Western blotting of XRCC6 expression in HCT116 and TRIM21 knockdown HCT116 cells treated with cycloheximide (CHX 100 µg/ml). G Western blotting of XRCC6 expression in SW480 and TRIM21 knockdown SW480 cells treated with cycloheximide (CHX 100 µg/ml). H Ubiquitination assay of XRCC6 performed in HCT116, CNOT7 knockdown HCT116 and CNOT7 and TRIM21 knockdown HCT116 with TRIM21 knockdown cells. I Ubiquitination assay of XRCC6 performed in SW480, CNOT7 knockdown SW480 and CNOT7 and TRIM21 knockdown SW480 with TRIM21 knockdown cells. J Ubiquitination assay of XRCC6 performed in 293 T cells co-transfected with HA-Ub, HA-Ub (K48 only), His-Ub (K63 only), His-XRCC6, siTRIM21, or empty vector plasmids. K Ubiquitination assay of XRCC6 performed in 293 T cells co-transfected with His-XRCC6, His-XRCC6 (K9R), His-XRCC6 (K238R), His-XRCC6 (K526R), HA-Ub, siTRIM21, or empty vector plasmids. L HCT116 and CNOT7 knockdown HCT116 cells were treated with Actinomycin D (5ug/ml) and mRNA was isolated at the indicated time points. Th RNA decay rate was calculated based on the remaining mRNA levels at each time point relative to time zero. M Analysis of P-body formation upon CNOT7 knockdown using an anti-Dcp1a rabbit antibody. Scale bar, 10 μm. N RIP-qPCR analysis of the association between CNOT7 and TRIM21 mRNA.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: Immunoprecipitation, Mass Spectrometry, Protein-Protein interactions, Co-Immunoprecipitation Assay, Western Blot, Expressing, Knockdown, Ubiquitin Proteomics, Transfection, Plasmid Preparation, Isolation

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A , B CCK8 assay showed that XRCC6 knockdown increased the radiation therapy sensitivity in CNOT7 overexpression HCT116 ( A ) and SW480 ( B ) cells. C – F Representative images and corresponding survival fraction curves of colony formation assays showed that XRCC6 knockdown increased radiotherapy sensitivity in HCT116 ( C , D ) and SW480 cells ( E , F ). G , H Representative images and quantitation of γH2AX positive nuclei in HCT116, CNOT7 overexpression HCT116 cells and CNOT7 overexpression XRCC6 knockdown HCT116 cells at different time points. I , J Representative images and quantitation of γH2AX positive nuclei in SW480, CNOT7 overexpression SW480 cells and CNOT7 overexpression XRCC6 knockdown SW480 cells at different time points. K , L Representative images of comet assay and quantitative analysis of tail moment in HCT116, CNOT7 overexpression HCT116 cells and CNOT7 overexpression XRCC6 knockdown HCT116 cells at different time points. M , N Representative images of comet assay and quantitative analysis of tail moment in SW480, CNOT7 overexpression SW480 cells and CNOT7 overexpression XRCC6 knockdown SW480 cells at different time points.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: CCK-8 Assay, Knockdown, Over Expression, Quantitation Assay, Single Cell Gel Electrophoresis

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: A Sensitivity curves for different doses of radiation treatment in HCT116 and CNOT7 knockdown HCT116 cells with or without STL127705 (30 µM) treatment. B Sensitivity curves for different doses of radiation treatment in SW480 and CNOT7 knockdown SW480 cells with or without STL127705 (30 µM) treatment. C Sensitivity curves for different concentrations of STL127705 in HCT116 and CNOT7 knockdown HCT116 cells with or without radiation. D Sensitivity curves for different concentrations of STL127705 in SW480 and CNOT7 knockdown SW480 cells with or without radiation. E Schematic diagram of construction and drug therapy for patient-derived tumor xenograft model. F Gross images of patient-derived tumor xenograft with or without radiotherapy and STL127705. G – I Tumor growth curves ( G ), tumor volume ( H ) and tumor weights ( I ) of patient-derived tumor xenograft with or without radiotherapy and STL127705. J Schematic diagram of drug therapy for HCT116 xenograft model. K Gross images of HCT116 and CNOT7 knockdown HCT116 cell-derived subcutaneous tumors with or without radiotherapy and STL127705. L – N Tumor growth curves ( L ), tumor volume ( M ) and tumor weights ( N ) of HCT116 and CNOT7 knockdown HCT116 cell-derived subcutaneous tumors with or without radiotherapy and STL127705.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: Knockdown, Derivative Assay

Journal: Cell Death & Disease

Article Title: CNOT7 facilitates radiation resistance in colorectal cancer through TRIM21/XRCC6-mediated non-homologous end joining repair

doi: 10.1038/s41419-025-08160-4

Figure Lengend Snippet: Schematic diagram of the regulatory mechanism by which CNOT7 promotes radiation resistance in colorectal cancer by regulating the XRCC6-mediated NHEJ DNA repair pathway through TRIM21.

Article Snippet: The antibodies were: CNOT7 (Proteintech, #14102-1-AP, RRID:AB_2245087), XRCC5 (Proteintech, #16389-1-AP, RRID:AB_2257509), Phospho-DNA-Pkcs (Ser2056) (CST, #68716, RRID:AB_2939025), XRCC6 (Proteintech, #10723-1-AP, RRID:AB_2218756), DNA-Pkcs (Proteintech, #19983-1-AP, RRID:AB_10642811), RAD50(Proteintech, #29390-1-AP, RRID:AB_2918289), NBS1(Proteintech, # 55025-1-AP, RRID:AB_10858928), Phospho-Histone H2A.X (Ser139) (20E3) (CST, #9718, RRID:AB_2118009), TRIM21 (Proteintech, #12108-1-AP, RRID:AB_2209469), RAD51(Proteintech, #14961-1-AP), 53BP1(Proteintech, # 20002-1-AP, RRID:AB_3085592), MRE11(Proteintech,#10744-1-AP,RRID:AB_2145118) and Ub (Proteintech, #10201-2-AP, RRID:AB_671515).

Techniques: