Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: RNA interference screen to identify novel regulators of sedDNA abundance. ( A ) HeLa cells were exposed to 20 J/m 2 UV, harvested at the indicated time points, and then sedDNA products of NER were visualized as described in the Materials and Methods section. The primary and secondary, partially degraded populations of sedDNAs are indicated. ( B ) Representative results from RNAi screen in which sedDNAs were isolated and detected in HeLa cells transfected with the indicated siRNA pool and exposed to 10 J/m 2 UV. ( C ) Quantitation of results (average and SEM) from five independent experiments performed as in (B). A one-sided t-test was used to determine whether knockdown of specific nucleases impacted sedDNA levels in comparison to the control transfection (* P < 0.05; ** P < 0.01; *** P < 0.001; **** P < 0.0001). ( D ) Quantitation of the percentage of primary and secondary sedDNAs in cells transfected with control and Trex1 siRNA. (D) Quantitation of the percentage of primary and secondary sedDNAs in cells transfected with control and Trex1 siRNA.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: Isolation, Transfection, Quantitation Assay, Knockdown, Comparison, Control

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Knockdown of Trex1 in human cells increases sedDNA levels in cells exposed to UV radiation and chemical carcinogens. ( A ) HeLa cells were transfected with in the indicated siRNA, exposed to 20 J/m 2 UV, and then harvested at the indicated time points for analysis of sedDNAs. ( B ) A375 melanoma cells were treated as in (A). ( C ) A375 cells were transfected as in (A) and exposed to the indicated fluences of UV radiation. Cells were harvested 1 h later for analysis of sedDNAs. (D, E) HeLa cells transfected as in (A) were exposed to either N -acetyoxy-2-acetylaminofluorene (AAF) or benzo[a]pyrene diol epoxide (BPDE) and then harvested at the indicated time points to detect the sedDNA products of NER. All graphs show the relative level of sedDNAs from at least three independent experiments. T -tests were used to compare the relative sedDNA abundance at each time point and UV dose.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: Knockdown, Transfection

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Knockdown of Trex1 increases the levels of (6-4)PP- and CPD-containing sedDNAs. ( A ) HeLa cells were treated as in Figure except that sedDNAs were immunoprecipitated with an antibody against (6-4)PPs. ( B ) Cells were processed as in (A) except that an anti-CPD antibody was used for immunoprecipitation. The graphs show the relative level of sedDNAs from at least three independent experiments.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: Knockdown, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Knockdown of Trex1 increases sedDNA association with the NER factors TFIIH and RPA. ( A ) HeLa cells were treated as in Figure except that cell lysates were immunoprecipitated with an anti-TFIIH antibody. ( B ) Cell lysates from cells treated as in (A) were immunoprecipitated with an anti-RNA antibody. ( C , D ) A375 cells were processes as for HeLa cells in (A) and (B). Graphs show the average level of TFIIH- and RPA-bound sedDNA from three independent experiments.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: Knockdown, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Overexpression of Trex1 decreases sedDNA abundance in UV-irradiated cells. ( A ) HeLa cells were transfected with expression vectors expressing the indicated fusion proteins, exposed to UV, and then harvested for analysis of sedDNAs. Quantitation of relative sedDNAs from five independent experiments. ( B , C ) Cells were processed as in (A) except that sedDNAs were purified and then immunoprecipitated with anti-(6-4)PP or anti-CPD antibodies.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: Over Expression, Irradiation, Transfection, Expressing, Quantitation Assay, Purification, Immunoprecipitation

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Recombinant TREX1 protein degrades the 3′ end of UV photoproduct-containing sedDNAs in vitro . ( A ) GFP-tagged wild-type and D18N mutant TREX1 proteins were purified from HeLa cells, separated by SDS-PAGE, and stained with Coomassie blue. The locations of the indicated proteins and antibody heavy (*) and light chains (**) are indicated. ( B ) In vitro nuclease assays in which the indicated recombinant proteins were mixed with sedDNAs purified from UV-irradiated cells and incubated for 1 h as described in the Materials and Methods section. Fractions of the reactions were analyzed for remaining sedDNAs (top) and for protein content by immunoblotting (bottom). ( C ) In vitro nuclease assays were performed as in (B) except that the reactions contained different amounts of each protein. ( D ) The reactions containing GFP-tagged wild-type TREX1 (0.16 ng/μl) were incubated with sedDNAs for the indicated time periods. All graphs show the average level of sedDNAs from at least three independent experiments. ( E ) Schematic of 30-nt-long model DNA substrate containing a single dipyrimidine sequence and a 5′ biotin. The DNA was exposed to UV radiation, subjected to immunoprecipitation with the indicated antibody, and then treated with TREX1 protein. ( F ) Analysis of model DNA substrates from (E) after digestion with recombinant TREX1. ( G ) Size analysis of (6-4)PP- and CPD-containing DNAs in (F) after digestion with TREX1. ( H ) Schematic of a second model DNA substrate lacking a 5′ biotin but treated as in (E, F) except that the DNA was 3′ end labeled with terminal transferase after digestion with TREX1. ( I ) Results from the treatment of the DNA substrate in (H) with TREX1. Note that control (non-irradiated) and UV-irradiated DNAs not subjected to immunoprecipitation with photoproduct antibodies were similarly digested with TREX1 and examined by urea PAGE and detection with HRP-streptavidin.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: Recombinant, In Vitro, Mutagenesis, Purification, SDS Page, Staining, Irradiation, Incubation, Western Blot, Sequencing, Immunoprecipitation, Labeling, Control

Journal: Nucleic Acids Research

Article Title: TREX1 degrades the 3′ end of the small DNA oligonucleotide products of nucleotide excision repair in human cells

doi: 10.1093/nar/gkac214

Figure Lengend Snippet: Schematic summarizing NER and the role of TREX1 in degrading the sedDNA products of the repair reaction. In response to exposure to UV radiation or other chemical carcinogens, the nucleotide excision repair machinery targets the damaged bases (indicated by T<>T) for removal via either XPC-dependent or RNA polymerase/CSA/CSB-dependent pathways. TFIIH unwinds the DNA around the lesion, which allows the XPF and XPG structure-specific nucleases to incise the damage strand 5′ and 3′ of the lesion, respectively. This dual incision releases the sedDNA from duplex DNA in complex with TFIIH. The gap can be filled in by DNA synthesis and ligation. The sedDNAs can be released from TFIIH and bind to RPA, and both the TFIIH- and RPA-sedDNA complexes are susceptible to digestion by TREX1, which degrades the nucleotides located 3′ the lesion. Because RPA-bound sedDNAs are known to be smaller on average than TFIIH-bound sedDNAs, we presume that additional 5′→3′ nucleases may partially degrade the sedDNA. Ultimately, the sedDNA likely undergoes additional degradation to smaller species. However, nothing is known about these latter steps of sedDNA processing.

Article Snippet: Cells were lysed in cold Triton X-100 lysis buffer (20 mM Tris–HCl, pH7.5, 150 mM NaCl, 1 mM EDTA, 1 mM EGTA and 1% Triton X-100) or RIPA buffer (25 mM Tris–HCl, pH 7.6, 150 mM NaCl, 1% NP-40, 1% sodium deoxycholate and 0.1% SDS) and analyzed by immunoblotting as previously described ( , ) using antibodies against TREX1 (Cell Signaling Technology), GAPDH (Cell Signaling Technology), GFP (Thermo Fisher Scientific), Flag epitope (Sigma).

Techniques: DNA Synthesis, Ligation

Journal: Signal Transduction and Targeted Therapy

Article Title: TRIM41 is required to innate antiviral response by polyubiquitinating BCL10 and recruiting NEMO

doi: 10.1038/s41392-021-00477-8

Figure Lengend Snippet: TRIM41 deficiency impairs innate response to pathogenic nucleic acid mimics and virus infections. a – d Trim41 +/+ or Trim41 –/– BMDM (1×10 5 cells per 24-well; a – c ) or BMDC (1 × 10 5 cells per 24-well; d ) were treated with liposome-packaged synthesized cytosolic nucleic acids sensor agonists for 4 h ( a ; 500 ng/ml 5′ppp-dsRNA for RIG-I, 500 ng/ml poly (I:C) for MDA5, 2 μg/ml HSV-60 or ISD for cGAS−STING), or indicated pathogens for 8 h ( b – d ) (MOI = 1 for VSV and SeV, MOI = 2 for LM, and MOI = 5 for HSV-1 or VACV). Amounts of IL-6, TNF, and IFNβ in supernatants were measured by ELISA ( a , b and d ), and the mRNA levels of ISGs were examined by Q-PCR ( c ). e 48 h after the transfection of Trim41 +/+ or Trim41 –/– BMDM cells with control or Trex1 - or Adar1 -specific siRNAs, the expression of indicated proteins were examined by Western blotting. f-h Cells in ( e ) were cultured in fresh medium for 48 h. Amounts of IL-6 and IFNβ in the supernatants were measured by ELISA ( f and g ), and the mRNA levels of Cxcl10 were examined by Q-PCR ( h ). Results are presented as mean ± SD of three biological replicates ( a – d and f – h ; one-way ANOVA followed by Bonferroni multiple comparison). One representative experiment of three is shown. ** P < 0.01; *** P < 0.001; **** P < 0.0001

Article Snippet: The siRNA duplexes specific for Adar1 (sc-37658) and Trex1 (sc-63158) were obtained from Santa Cruz Biotechnology (Dallas, TX).

Techniques: Virus, Synthesized, Enzyme-linked Immunosorbent Assay, Transfection, Control, Expressing, Western Blot, Cell Culture, Comparison

Journal: aging and disease

Article Title: Age-Associated Activation of the cGAS-STING Pathway and Impairment of DNA Damage Repair in Human Primary Alveolar Type II Cells

doi: 10.14336/ad.2024.1175

Figure Lengend Snippet: Figure 2. Increased DNA damage, TREX1, and 8-OHdG levels in ATII cells in older smokers. BALF, ATII cells, and lung tissue were obtained from younger and older non-smokers (NS) and smokers (SM). ssDNA (A) and dsDNA (B) levels in BALF. (C) DNA damage was analyzed in ATII cells by comet assay (scale bar – 5 µm). Quantification of the Olive tail moment is shown. (D) ATII cells were treated with etoposide for 24h to induce DNA damage. (E) Recovery was determined using drug-free media for 24h. (F) dsDNA cytosolic levels were analyzed in ATII cells identified using SP-C antibody (scale bar – 5 µm). Quantification of fluorescence intensity is also shown. (G) TREX1 expression in ATII cells was detected using a proSP-C antibody (scale bar – 5 µm). (H) 8-OHdG levels were analyzed in ATII cells identified using a proSP-C antibody (scale bar – 5 µm). Data are expressed as means±SEM.*p<0.05; **p<0.01 (Kruskal-Wallis test).

Article Snippet: Aging and Disease • Volume 17, Number 1, February 2026 4 Paraffin-embedded lung tissue sections were incubated with the following antibodies obtained from Santa Cruz Biotechnology (Dallas, TX): p-ATM (sc-47739), SP-C (sc-518029), dsDNA (sc-58749), TREX1 (sc-133112), 8- OHdG (sc-393871), cGAS (sc-515802) or purchased from other manufacturers: proSP-C (AB3786; Millipore, Burlington, MA), SP-A (NBP2-12928, Novus Biologicals, Littleton, CO), 4-HNE (MAB3249; R&D Systems, Minneapolis, MN), STING (PA523381; Invitrogen Corp., Carlsbad, CA).

Techniques: Single Cell Gel Electrophoresis, Fluorescence, Expressing