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: The FASEB Journal

Article Title: Chronic gestational hypoxia accelerates ovarian aging and lowers ovarian reserve in next-generation adult rats

doi: 10.1096/fj.201802772R

Figure Lengend Snippet: Primer sequences and product size

Article Snippet: Detection steps used the following primary antibodies; 8-oxoguanine DNA glycosylase (OGG1; NB100-106, 1:1000; Novus Biologicals, Centennial, CO, USA), NTH1 (11154-1-AP, 1:1000; Proteintech, Rosemont, IL, USA), hypoxia-inducible factor 1α (HIF1α; Ab51608, 1:1000; Abcam, Cambridge, MA, USA), catalase (Ab1877-10, 1:10,000; Abcam), MnSOD (06–984; Upstate, Watford, United Kingdom), copper/zinc superoxide dismutase (CuZnSOD; 10269-1-AP, 1:1000; Proteintech), NADPH oxidase 1 (GP91 phox ; 19013-1-AP; Proteintech), neutrophil cytosolic factor 2 (P67 phox ; 15551-1-AP, 1:1000; Proteintech), xanthine oxidase (XO; SC-20991, 1:200; Santa Cruz Biotechnology, Dallas, TX, USA), tumor protein 53 (P53; MAB1355, 1:1000; R&D Systems, Minneapolis, MN, USA), cyclin-dependent kinase inhibitor 2A (P16 ink ; Ab189034, 1:1000; Abcam), KU70 (10723-1-AP, 1:1000; Proteintech), KU80 (NB100-508, 1:1000; Novus Biologicals).

Techniques: Sequencing

Journal: International Journal of Molecular Sciences

Article Title: Bioengineered Skin from a Platelet-Derived Hydrogel Repairs Full Thickness Wounds in a Pre-Clinical Mouse Model

doi: 10.3390/ijms26209988

Figure Lengend Snippet: Full thickness wound repair by engineered skin grafting in athymic nude mice . ( A ) Representative images of the wounds grafted with PG-HSE, BTM-HSE, or native skin autografts on day of grafting (day 0) and post-grafting (day 14). ( B ) Representative microscopic images of day 14 grafts analysed by immunofluorescence, using a human specific antibody against involucrin. PG-HSE pre-grafting (scale 100 µm); PG-HSE graft (scale 1 mm); BTM-HSE pre-grafting (scale 100 µm); BTM-HSE graft (scale 1 mm); autograft pre-grafting (scale 100 µm; autograft (scale 1 mm); and human native skin (scale 1 mm). Zoomed images scale bar = 50 µm. Orange arrows indicate the graft edge 2 weeks post-grafting and red arrows indicate the initial wound edge. ( C ) Presence of human involucrin in grafts is presented as integrated density (IntDen), that is, sum of pixel value/signal intensity within a selected region of interest calculated using FIJI software version 1.54p. ( D ) Human-specific Ku80 marker was detected by immunofluorescence on day 14 post-grafting and % Ku80 positive cells were counted using Nikon NIS-Elements Analysis software (version 5.5). Data were analysed using ordinary one-way ANOVA. Values in ( C , D ) represent mean values +/− SEM in each group (* = p ≤ 0.05, ** = p ≤ 0.01, n = 4–5 per group). PG-HSE, platelet-derived human skin equivalent; BTM-HSE, NovoSorb biodegradable temporising matrix human skin equivalent; autograft, and autologous full thickness skin graft.

Article Snippet: Slides were blocked as above and incubated with primary antibodies: rabbit anti-human involucrin (1:100, Cat. No. ab234403, Abcam), rabbit anti-human Ku80 (1:50, Cat. No. 214745, Biorbyt, Durham, NC, USA), or rabbit anti-human CK5 (1:250, Cat. No. 905504, BioLegend, San Diego, CA, USA ) overnight.

Techniques: Immunofluorescence, Software, Marker, Derivative Assay

Journal: Nature Communications

Article Title: Profiling ubiquitin signalling with UBIMAX reveals DNA damage- and SCF β-Trcp1 -dependent ubiquitylation of the actin-organizing protein Dbn1

doi: 10.1038/s41467-023-43873-0

Figure Lengend Snippet: a Experimental outline for the UBIMAX experiment profiling ubiquitylated proteins in response to DPC-containing substrates. Extracts were untreated or supplemented with ubiquitin E1 inhibitor (“Ub E1i”) prior to addition of His 6 -Ubiquitin (“His 6 -Ub”). Reactions were initiated by addition of buffer (“no DNA”), undamaged plasmid DNA (“DNA”), plasmids carrying the M.HpaII protein crosslinked at a single-stranded DNA gap (“ssDNA-DPC”), or plasmids carrying the Flp protein crosslinked at a single-strand break (“SSB-DPC”). Reactions were performed in independent reaction triplicates. Samples were harvested at 30 min and subjected to the UBIMAX workflow outlined in Fig. . b , c Volcano plot analysis comparing ubiquitylated proteins enriched from ssDNA-DPC ( b ) or SSB-DPC ( c ) versus DNA-treated samples. Pink/orange and blue dots indicate significantly enriched and -depleted ubiquitylated proteins. Significance was determined by two-tailed Student’s t -test, with permutation-based FDR-control with s0 = 0.1 and 2500 rounds of randomization, to ensure an FDR ≤ 0.05. Ubiquitylated proteins with FDR ≤ 0.01 are labelled. d – i Abundance of Ku80 ( d ) Ku70 ( e ) Mre11 ( f ) Rpa1 ( g ) Chfr ( h ) and Dbn1 ( i ) across ubiquitin target enriched samples of the UBIMAX experiments profiling protein ubiquitylation in response to DSBs (Fig. ) and DPCs (a). Horizontal lines indicate the median and significance was determined by one-way ANOVA with Dunnett’s multiple comparisons test for all conditions against undamaged DNA with a cut-off of p -value ≤ 0.01 and with those indicated of p -values ˂ 0.0001. n = 3 independent reaction replicates for DSB and DPC conditions and n = 7 for no DNA and DNA conditions. a.u., arbitrary units. j Extracts were untreated or supplemented with ubiquitin E1 inhibitor prior to initiation of reactions by addition of undamaged plasmid DNA (“DNA”), linearized plasmid DNA (“DSB”), or plasmids carrying a DPC at a ssDNA gap (“ssDNA-DPC”). Samples were analysed by western blot at the indicated times. *unspecific band. Source data are provided as a Source Data file.

Article Snippet: The following antibodies were raised against the indicated peptides derived from Xenopus laevis proteins (New England Peptide, now Biosynth): Dbn1 (Ac-CWDSDPVMEEEEEEEEGGGFGESA-OH, 1:1000), Ku80 (CMEDEGDVDDLLDMM, 1:1000), Cul1 (H2N-MSSNRSQNPHGLKQIGLDQC-amide, 1:2500), Fbxl12 (Ac-CRGIDELKKSLPNSKVTN-OH, 1:2500), Psa3 (Ac-CKYAKESLEEEDDSDDDNM-OH, 1:5000), β-Trcp1-INT (Ac-GQYLFKNKPPDGKTPPNSC-amide), β-Trcp1-N (H2N-MEGFSSSLQPPTASEREDC-amide), and Dbn1-pS609/611 (Ac-CSEGYF(pS)Q(pS)QDED-amide, 1:2500).

Techniques: Ubiquitin Proteomics, Plasmid Preparation, Two Tailed Test, Control, Western Blot