Journal: The Journal of Biological Chemistry

Article Title: TOX High-Mobility Group Box Family Member 4 promotes DNA double-strand break repair via nonhomologous end joining

doi: 10.1016/j.jbc.2025.110174

Figure Lengend Snippet: TOX4 binds KU proteins and mediates DNA-PKcs activation. A , TOX4 immunoprecipitation (IP) was performed in HeLa cell lysates, and the IP project was subjected to mass spectrometric identification. KU70 and KU80 were among the identified proteins, as shown with the number of peptides. A control IP using IgG did not recover any peptides of TOX4, KU70, or KU80. The specific binding proteins of TOX4, identified in this proteomic study, are shown in . B , KU80 IP was performed in HeLa cell lysates. The lysate input at 20%, control (ctr) IP with blank beads and KU80 IP products were analyzed by immunoblotting for TOX4, KU70, and KU80. C , GFP-TOX4 was expressed in HeLa cells, and GFP IP was performed. The lysate input at 20%, control IP, and GFP IP products was analyzed by immunoblotting for TOX4, KU80, and β-actin. D , TOX4 IP was performed in HepG2 cell lysates. The lysate input at 20% control (ctr) IP with blank beads and TOX4 IP products were analyzed by immunoblotting for TOX4, KU70, and KU80. E , the schematic diagram of TOX4 mutants generated in this study. F , full-length and two segments of TOX4 (ΔC: aa 1–591; C: aa 591–621) were tagged with GFP and expressed in HeLa cells. GFP IP was performed in cells expressing these proteins or control cells. The lysate input at 20% and GFP IP products were analyzed by immunoblotting for GFP, β-actin, and KU80. G , HeLa cells were treated with TOX4 siRNA and doxorubicin (DOX) (5 μM) for 4 h, as indicated. The cell lysates were analyzed by immunoblotting for phospho-DNA-PKcs Ser-2056, DNA-PKcs, Chk2 phospho-Thr-68, TOX4, and β-actin. H , cells were analyzed as in G . Corresponding densitometric measurement of phospho-DNA-PKcs Ser-2056 corrected by β-actin was shown. Statistical significance was analyzed using an unpaired two-tailed Student t test (∗∗∗ p < 0.001). I , HeLa cells were transfected with TOX4-ΔC segment and treated with DOX, as indicated. The cell lysates were analyzed by immunoblotting for DNA-PK phospho-Ser-2056, Smc1 phospho-Ser-957, TOX4, and β-actin. J , corresponding densitometric analyses of phosphor-DNA-PKcs Ser-2056/β-actin, in I , are shown. Statistical significance was analyzed using an unpaired two-tailed Student t test (∗∗∗ p < 0.001). K , HeLa cells were transfected with TOX4 siRNA, TOX4-ΔC, and TOX4-WT, and treated with DOX, as indicated. The cell lysates were analyzed by immunoblotting for DNA-PKcs phospho-Ser-2056, TOX4, and GAPDH. L , corresponding densitometric analyses of phospho-DNA-PKcs Ser-2056/β-actin in J are shown. Statistical significance was determined using an unpaired two-tailed Student t test (∗∗ p < 0.01, ns: p > 0.05). DNA-PKcs, DNA-dependent protein kinase catalytic subunit; TOX4, Thymocyte Selection–Associated High-Mobility Group Box Family Member 4.

Article Snippet: SDS-PAGE and IB were carried out as previously described , using the following antibodies: KU80 (A302-627A), γ-H2AX (A300-081A), PNUTS (A300-439A), and Smc1 phosphoS957 (A304-147A) antibodies from Bethyl Laboratories; GFP (sc-9996) and KU70 (sc-56129) antibodies from Santa Cruz Biotechnology; TOX4 (ab272576), Chk2 phospho-T387 (ab195783), PAR (ab14459), DNA-PKcs (ab70250), DNA-PKcs phospho-S2056 (ab18192) antibodies from Abcam; β-actin (#4970), tubulin (#2144), caspase-3 (#9662), and Chk2 phospho-T68 (#2661) antibodies from Cell Signaling Technology.

Techniques: Activation Assay, Immunoprecipitation, Control, Binding Assay, Western Blot, Generated, Expressing, Two Tailed Test, Transfection, Selection

Journal: The Journal of Biological Chemistry

Article Title: TOX High-Mobility Group Box Family Member 4 promotes DNA double-strand break repair via nonhomologous end joining

doi: 10.1016/j.jbc.2025.110174

Figure Lengend Snippet: TOX4 and PNUTS coordinate in NHEJ. A , HeLa cells were treated with siRNA targeting TOX4 or PNUTS, as indicated, for 24 h. The cell lysates were analyzed by immunoblotting for γ-H2AX, TOX4, PNUTS, and tubulin. B , NHEJ repair efficiency was measured in HeLa cells using the linearized GFP vector, as in N . Cells were treated with or without siRNA targeting TOX4, PNUTS, or KU80, as indicated. Immunoblots of GFP, TOX4, PNUTS, and β-actin are shown. C , Ku80 IP was performed in HeLa cells treated with control or TOX4 siRNA. The lysate input at 20%, control (ctr) IP with blank beads, and KU80 IP products were analyzed by immunoblotting for PNUTS, TOX4, KU80, and β-actin. NHEJ, nonhomologous end joining; PNUTS, phosphatase 1 nuclear targeting subunit; TOX4, Thymocyte Selection–Associated High-Mobility Group Box Family Member 4.

Article Snippet: SDS-PAGE and IB were carried out as previously described , using the following antibodies: KU80 (A302-627A), γ-H2AX (A300-081A), PNUTS (A300-439A), and Smc1 phosphoS957 (A304-147A) antibodies from Bethyl Laboratories; GFP (sc-9996) and KU70 (sc-56129) antibodies from Santa Cruz Biotechnology; TOX4 (ab272576), Chk2 phospho-T387 (ab195783), PAR (ab14459), DNA-PKcs (ab70250), DNA-PKcs phospho-S2056 (ab18192) antibodies from Abcam; β-actin (#4970), tubulin (#2144), caspase-3 (#9662), and Chk2 phospho-T68 (#2661) antibodies from Cell Signaling Technology.

Techniques: Western Blot, Plasmid Preparation, Control, Selection

Journal: STAR Protocols

Article Title: Laser microirradiation as a tool to investigate the role of liquid-liquid phase separation in DNA damage repair

doi: 10.1016/j.xpro.2022.101146

Figure Lengend Snippet:

Article Snippet: KU80-GFP plasmid , , Addgene Cat#46958.

Techniques: Recombinant, Plasmid Preparation, Clone Assay, Software, Pore Size, Sterility, Microscopy, Fluorescence