Journal: Nucleic acids research

Article Title: Monitoring eukaryotic and bacterial UDG repair activity with DNA-multifluorophore sensors.

doi: 10.1093/nar/gkt309

Figure Lengend Snippet: Figure 4. In vitro responses of probes 2, 4 and control 6 to human UNG2 enzyme. (A) Time courses of response of probes at 480 nm. (B) Spectral changes in response of probe 2 over the same time course. Excitation = 340 nm. Condition: [hUNG2] = 9.7 mg/ml (90 nM); [probe] = 4 mM; 37C, excitation 340 nm.

Article Snippet: When the fluorescence intensity was stable, the UDG (New England Biolabs), human UNG2 (Origene) or human SMUG1 (Origene) was added to the solution.

Techniques: In Vitro, Control

Journal: Nature Communications

Article Title: BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

doi: 10.1038/s41467-025-64578-6

Figure Lengend Snippet: a Schematic of the full-length UNG2 protein, the probe (3XFLAG-ΔUNG), and the method used for U-ssDNA detection. b Western blot analysis of BRCA2 in cells transfected with two independent BRCA2 siRNAs (siB2#5 or siB2#8); vinculin serves as a loading control. c , f Representative images of U-ssDNA accumulation in U2OS cells transfected with the indicated siRNAs. Cells were treated with cisplatin (50 µM, 1 h; collected 24 h post recovery) or HU (2 mM, 30 h) and stained with ΔUNG probe under nondenaturing conditions. d , e , g , h Quantification of U-ssDNA foci/cell for ( c , f ). Foci were analyzed in Image J; 100–200 cells per replicate. SuperPlots of three independent biological replicates ( n = 3 are plotted. Highlighted shapes (circle, square, and triangle) represent the average of each replicate, and black lines represent mean ± SD. i U-ssDNA accumulation in PEO1/PEOC4 or CAPAN-1/C2-12 cells. Cells were not-treated (NT) or treated with cisplatin and HU as in ( c , f ). j , k Quantification of U-ssDNA intensity (arbitrary units) per cell detected with ΔUNG probe in PEO1/PEOC4 (j) or CAPAN-1/C2-12 ( k ) cells; > 200 cells were analyzed per replicate. SuperPlots are plotted as described for ( d ). l Western blot analysis of BRCA2 in U2OS cells containing a doxycycline (DOX)-inducible shRNA against BRCA2. Cells were treated with or without doxycycline (10 µg/mL, 72 h). LaminB1 is the loading control. m Schematic of doxycycline, cisplatin, and HU treatment. n U-ssDNA foci in U2OS cells with DOX-inducible BRCA2 shRNA. Cells were treated with doxycycline for 72 h prior to cisplatin (50 µM, 1 h; 24 h recovery) or HU (2 mM, 30 h). o Quantification of U-ssDNA foci. SuperPlots are plotted as described for ( d ). Statistical significance for all the sets of three independent biological replicates ( n = 3) analyzed above was determined using a repeated-measurement model followed by two-tailed multiple comparisons with a Bonferroni post hoc test. **** p ≤ 0.0001; * p ≤ 0.05; ns, not significant. In all images, the scale bar represents 20 µm. Western blot images presented here are representative of three or more western blots with similar results. Source data are provided as a Source Data file.

Article Snippet: UNG2 CRISPR-Cas9 Double Nickase plasmid was purchased from Santa Cruz Biotechnology (SCBT, (sc-403189-NIC) and transfected into U2OS cells using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Western Blot, Transfection, Control, Staining, shRNA, Two Tailed Test

Journal: Nature Communications

Article Title: BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

doi: 10.1038/s41467-025-64578-6

Figure Lengend Snippet: a Western blot of BRCA2 in U2OS cells transfected with two independent BRCA2 siRNAs (siB2#5 or siB2#8); vinculin is the loading control. b Dot blot detection of abasic sites (AP-DNA) in U2OS cells transfected with siLuc (control) or siBRCA2 siRNAs and then untreated (NT) or treated with cisplatin (50 µM, 1 h, 24 h recovery) or HU (2 mM, 30 h). Methylene blue served as the loading control. One of four independent experiments is shown. c Quantification of AP-DNA intensity normalized to methylene blue. Fluorescence intensity is plotted as arbitrary (arb.) units. Data are mean ± SD, n = 4 independent experiments. Statistical significance was determined by an unpaired, two-tailed Student’s t test. **** p ≤ 0.0001; ns = not significant. d , e Quantification of AP-DNA intensity in PEO1/PEOC4 (d) or CAPAN-1/C2-12 ( e ) cells, treated as in ( b ). Mean ± SD of n = 4 independent experiments are plotted. Statistical significance was determined by an unpaired, two-tailed Student’s t test. *** p ≤ 0.001; ** p ≤ 0.01; ns = not significant. f Western blot of BRCA2 and BRCA1 after siRNA; LaminB1 is a control. g Quantification of AP-DNA intensity in cells transfected with the indicated siRNAs, treated as in ( b ). Data from mean ± SD, n = 4 independent experiments. Statistical significance determined by un-paired two-tailed Student’s t test. **** p ≤ 0.0001; ** p ≤ 0.01; ns=not significant. h Quantification of AP-DNA intensity in BRCA1 mutant UWB1.289 cells after cisplatin or HU. Data from mean ± SD, n = 4 independent experiments. Statistical significance was determined as described for ( g ). i Western blot of BRCA2 and UNG2 after siRNA; α-tubulin is the loading control. j Quantification of AP-DNA intensity in U2OS cells transfected with indicated siRNAs, treated as in ( b ). Data are mean ± SD of n = 4 independent experiments. Statistical significance was determined as described for ( g ). k Western blot of BRCA2 and UNG2 in U2OS or UNG2-KO U2OS cells after siRNA. l Quantification of AP-DNA intensity after treatment with cisplatin and HU as described in ( b ). Data from mean ± SD of n = 7 independent experiments. Statistical significance was determined as described in ( g ). Western blot images presented represent ≥ 3 blots with similar results. Source data are provided as a Source Data file.

Article Snippet: UNG2 CRISPR-Cas9 Double Nickase plasmid was purchased from Santa Cruz Biotechnology (SCBT, (sc-403189-NIC) and transfected into U2OS cells using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Western Blot, Transfection, Control, Dot Blot, Fluorescence, Two Tailed Test, Mutagenesis

Journal: Nature Communications

Article Title: BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

doi: 10.1038/s41467-025-64578-6

Figure Lengend Snippet: a , b Western blots of BRCA2 with UNG2 ( a ) or APE1 ( b ) in U2OS cells transfected with indicated siRNAs; α-tubulin ( a ) or GAPDH ( b ) is the loading control. c Representative images of 53BP1 immunostaining in U2OS cells transfected with the indicated siRNAs, treated with HU (5 mM, 4 h), followed by 18 h recovery. Scale bar represents 20 µm. d , e Quantification of 53BP1-positive cells showing S-phase specific foci pattern (see Supplementary Fig. ). Data are mean ± SD of n = 3 independent experiments, each with two technical replicates (> 200 cells/replicate). f Western blot of BRCA2 and GFP (UNG2-EYFP) in U2OS or UNG2-KO U2OS cells transfected with the indicated siRNAs. Samples collected 72 h post siRNA transfection. Vinculin is the loading control. g Representative images of 53BP1 immunostaining in cells treated as in ( f ). Scale bar represents 20 µm. h Quantification of 53BP1 positive cells with S-phase specific foci pattern. Data are mean ± SD of n = 3 independent experiments, each with two technical replicates (> 200 cells/replicate). i Treatment schematic for cisplatin or HU treatment in U2OS cells analyzed for micronuclei formation. j , k Representative images of micronuclei in U2OS cells transfected with the indicated siRNAs and treated with cisplatin and HU. Scale bar represents 20 µm. l , m Quantification of micronuclei positive cells. Data are mean ± SD of n = 3 independent experiments with two technical replicates (150–200 cells/replicate). n Representative images of micronuclei in U2OS or UNG2-KO U2OS cells transfected with the indicated siRNAs, followed by transfection with vector or UNG2-EYFP. Cells were treated with cisplatin and HU as in ( i ). Scale bar represents 20 µm. o Quantification of micronuclei positive cells for ( n ). Data are shown as mean ± SD of n = 3 independent experiments with two technical replicates each. For all the graphs presented here, statistical significance was determined by an un-paired two-tailed Student’s t test. **** p ≤ 0.0001; *** p ≤ 0.001; ** p ≤ 0.01; * p ≤ 0.05; ns: not significant p > 0.05. Western blot images presented here are representative of three or more western blots with similar results. Source data are provided as a Source Data file.

Article Snippet: UNG2 CRISPR-Cas9 Double Nickase plasmid was purchased from Santa Cruz Biotechnology (SCBT, (sc-403189-NIC) and transfected into U2OS cells using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Western Blot, Transfection, Control, Immunostaining, Plasmid Preparation, Two Tailed Test

Journal: Nature Communications

Article Title: BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

doi: 10.1038/s41467-025-64578-6

Figure Lengend Snippet: a Western blot of BRCA2, APE1, and UNG2 in U2OS cells transfected with indicated siRNAs; vinculin is the loading control. b Left: Representative images of neutral comet in U2OS cells transfected with the indicated siRNAs and treated with cisplatin or HU. Scale bar represents 50 µm. Right: Quantification of average tail moment. For each experiment, 100–150 comets were analyzed by ImageJ/OpenComet. Data are mean ± SD of n = 3 independent experiments. Statistical significance was determined by an un-paired two-tailed Student’s t test. *** p ≤ 0.001; ** p ≤ 0.01; ns, not significant. c , f Western blots of BRCA2 and UNG2 in U2OS ( c ) or UNG2-KO U2OS ( f ) cells transfected with the indicated siRNAs; α-tubulin and vinculin are the loading control. d , e , g , h CellTiter-Glo survival assays of U2OS or U2OS UNG2 KO cells transfected with the indicated siRNAs and treated with HU or cisplatin. Error bars represent SD between triplicates. i Western blot of BRCA2 and APE1 in U2OS cells transfected with indicated siRNAs; GAPDH is the loading control. j , k CellTiter-Glo survival assay with U2OS cells transfected with the indicated siRNAs and treated with HU ( j ) or cisplatin ( k ). Error bars represent SD between triplicates. l Kaplan-Meier curve of 5-year overall survival for ovarian patients with BRCA2 mutant tumors (TCGA dataset). The patients are stratified by median APE1 expression. Mutations associated with the patient data are listed in the Source Data file. Western blot images presented here are representative of three or more western blots with similar results. Source data are provided as a Source Data file.

Article Snippet: UNG2 CRISPR-Cas9 Double Nickase plasmid was purchased from Santa Cruz Biotechnology (SCBT, (sc-403189-NIC) and transfected into U2OS cells using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Western Blot, Transfection, Control, Two Tailed Test, Clonogenic Cell Survival Assay, Mutagenesis, Expressing

Journal: Nature Communications

Article Title: BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

doi: 10.1038/s41467-025-64578-6

Figure Lengend Snippet: a Western blot of BRCA2 in U2OS cells transfected with the indicated siRNAs. GAPDH is the loading control. b Representative images of proximity ligation assay (PLA) using anti-ssDNA and anti-actin antibodies in U2OS cells transfected with the indicated siRNAs and treated with cisplatin (50 µM, 1 h, 24 h) or HU (2 mM, 30 h). Scale bar represents 20 µm. c Quantification of PLA puncta/cell from ( b ). SuperPlots of three independent experiments are presented ( n = 3). The number of PLA puncta/cell was analyzed through Image J; 100–200 cells/replicate. Each highlighted shape (circle, triangle, or square) represents the average of each replicate, with the black lines representing the mean ± SD. d Western blot of BRCA2, A3B, APE1, and UNG2 in U2OS cells used in ( e , f ). e Representative PLA images in U2OS cells transfected with the indicated siRNAs and then treated with cisplatin or HU. Scale bar represents 20 µm. f Quantification of PLA puncta/cell for ( e ). SuperPlots of three independent experiments; 100–200 cells/replicate are plotted as described in ( c ). g , h Western blots of BRCA2 and A3B in nuclear extracts from wildtype or UNG2 KO U2OS cells transfected with the indicated siRNAs and treated with cisplatin or HU. LaminB1 is the loading control. Fold change compared to siLuc untreated (NT) is determined by first normalizing to the loading control. i Representative U-ssDNA images in U2OS or UNG2 KO U2OS cells transfected with the indicated siRNAs and treated with cisplatin or HU. Scale bar represents 50 µM. j Quantification of U-ssDNA foci. SuperPlots of three independent experiments are plotted as described in ( c ). Statistical significance for all charts presented here was determined by using a repeated measurement model followed by two-tailed multiple comparisons with a Bonferroni Post hoc test. **** p ≤ 0.0001; ** p ≤ 0.01. Western blot images presented here are representative of three or more western blots with similar results. Source data are provided as a Source Data file.

Article Snippet: UNG2 CRISPR-Cas9 Double Nickase plasmid was purchased from Santa Cruz Biotechnology (SCBT, (sc-403189-NIC) and transfected into U2OS cells using Lipofectamine 3000 reagent (Invitrogen).

Techniques: Western Blot, Transfection, Control, Proximity Ligation Assay, Two Tailed Test

Journal: Nature Communications

Article Title: BRCA2 deficiency and replication stress drive APOBEC3-Mediated genomic instability

doi: 10.1038/s41467-025-64578-6

Figure Lengend Snippet: Replication stress leads to stalled fork collapse in an A3B-, UNG2-, and APE1-dependent manner, resulting in the generation of ssDNA fragments in the cytoplasm which go on to activate NF-κB. This, in turn, drives APOBEC3B upregulation and establishes a positive self-reinforcing loop that promotes mutagenic evolution and genomic instability in BRCA2-deficient cells.

Article Snippet: UNG2 CRISPR-Cas9 Double Nickase plasmid was purchased from Santa Cruz Biotechnology (SCBT, (sc-403189-NIC) and transfected into U2OS cells using Lipofectamine 3000 reagent (Invitrogen).

Techniques: