Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 polymorphisms and the risk of CIN III and cervical carcinoma

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Control

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 and rs2029167 polymorphisms with the risk of HR-HPV positive cervical carcinoma and CIN III

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Control

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 polymorphisms and the risk for CIN III and cervical carcinoma stratified by the sexual, reproductive history

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Infection

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs2029167 polymorphisms and the risk for CIN and cervical carcinoma stratified by the sexual, reproductive history

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Infection

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: Association between SMUG1 rs3087404 and rs2029167 polymorphisms and the risk for cervical carcinoma stratified by clinical pathological characteristics

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques:

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: SMUG1 mRNA expression in CSCCs with different genotypes of rs3087404 and rs2029167 (qPCR).

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Expressing

Journal: Journal of Cancer

Article Title: Association of SMUG1 SNPs in Intron Region and Linkage Disequilibrium with Occurrence of Cervical Carcinoma and HPV Infection in Chinese Population

doi: 10.7150/jca.27103

Figure Lengend Snippet: SMUG1 protein expression in CSCCs with different genotypes of rs3087404 and rs2029167 (Western Blot) (A) AA: rs3087404 genotype is AA; AG: rs3087404 genotype is AG; GG: rs3087404 genotype is GG; (B) AA: rs2029167 genotype is AA; AG: rs2029167 genotype is AG; GG: rs2029167 genotype is GG

Article Snippet: After blocking with 5% non-fat milk for 1h, PVDF membrane was incubated with primary mouse monoclonal antibodies: SMUG1 (1:2000) purchased from NOVUS Biologicals (Cat No. H00023583-M07) and GAPDH (1:5000) purchased from Proteintech(Cat No. Cat.60004-1-Ig) for 4°C overnight, then were washed with TBS containing 0.05%Tween-20 for three times, followed by a 1h incubation with an HRP-conjugated secondary antibody (1:5000).

Techniques: Expressing, Western Blot

Journal: Breast cancer research and treatment

Article Title: Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1) deficiency is linked to aggressive breast cancer and predicts response to adjuvant therapy.

doi: 10.1007/s10549-013-2769-6

Figure Lengend Snippet: Fig. 1 SMUG1 mRNA expression in breast cancer. a Kaplan–Meier curves showing breast cancer-specific survival (BCSS). b Disease-free survival (DFS) in the training set

Article Snippet: Primary antibodies used was an anti-SMUG1 goat polyclonal antibody (catalogue no: AP08884PU-N, Acris Antibody GmbH, Germany).

Techniques: Expressing

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 2. UV-DDB stimulates SMUG1 on uridine containing DNA. (A) Schematic representation of the DNA substrate containing dU:dA or dU:dG pair (left) or 5-hmdU:dA or 5-hmdU:dG pair (right) and the proposed reaction scheme. (B, D, F, H) Stimulation of SMUG1 excision kinetics by UV-DDB. SMUG1 (2.5 nM) was incubated with dsDNA (50 nM) containing dU:dA pair (B), dU:dG pair (D), 5-hmdU:dA pair (F), 5-hmdU:dG pair (H) in the absence (−) or presence (+) of UV-DDB (50 nM) at 37◦C. Aliquots were withdrawn at each time point and analyzed on a 10% denaturing polyacrylamide gel. Positions of the un-cleaved full-length substrate and excised product are indicated by arrows. (C, E, G, I) Quantification of the stimulation of SMUG1 excision kinetics by UV-DDB in (B, D, F and H, respectively). Excision product formation was quantified using ImageJ software. The excision percentage was plotted as mean ± SEM from three independent experiments, each run on duplicate gels.

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques: Incubation, Software

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 3. Single-molecule analysis reveals that UV-DDB stimulates SMUG1 by facilitated dissociation. (A) Experiments design of DNA tightrope assay to study UV-DDB induced dissociation of SMUG1. (top) Long DNA substrates with defined abasic sites (THF) every 2 kb are suspended between silica beads (bottom, left) His-tagged SMUG1 is labeled with primary mouse-anti-His antibody and secondary goat-anti-mouse antibody conjugated to a 605 nm Qdot, (bottom, right) unlabeled UV-DDB. (B) Stacked bar graph showing the fraction of motile (green) versus stationary (white) and persistent (solid) versus dissociating (diagonal lines) particles of 605 Qdot labeled SMUG1 in the absence (–) or presence (+) of unlabeled 1× UV-DDB on abasic (THF) DNA during 300s observation. (****, P < 0.0001 by 2 test). (C) Effects of UV-DDB on the binding lifetimes of SMUG1–DNA complexes. Data plotted as the mean ± SEM from three independent experiments. For each condition, CRTD (cumulative residence time distribution) is fit to a single exponential decay function to obtain the half-life. (D) Anomalous diffusion exponent () versus diffusion coefficient (log10D) plotted for SMUG1 (black-filled circles) and SMUG1 with 1× UV-DDB (red-filled circles). (E) Box and whisker plot (10–90 percentile) of left, the Anomalous diffusion exponent () and right, the diffusion coefficient (log10D) calculated for SMUG1 only (n = 50 particles) and SMUG1 with 1× UV-DDB (n = 24 particles) phases on long DNA substrates with defined abasic sites (THF) every 2 kb. +, sample mean, ** P < 0.01, ns, non-specific by two-tailed Student’s t-test (F) Image of 605 Qdot- labeled SMUG1 (green) on abasic (THF) tightrope suspended between beads in the 1× presence of unlabeled UV-DDB. Scale bar represents 2.5 m. Arrows point to dissociated SMUG1 particles. Horizontal and vertical scale bars represent 50 s and 2 kb, respectively. (G) Image of 605Q dot-labeled SMUG1 (green) on abasic (THF) tightrope suspended between beads in the 1× presence of unlabeled UV-DDB. Scale bar represents 2.5 m. Arrow points to the dissociated SMUG1 particle. (H) Kymograph of 605 Qdot-labeled SMUG1 (green) with non-motile or motile dissociated particles (F). (I) Kymograph of 605 Qdot-labeled SMUG1 (green) with dissociated particle (G). Horizontal and vertical scale bars represent 50 s and 2 kb, respectively.

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques: Labeling, Binding Assay, Diffusion-based Assay, Whisker Assay, Two Tailed Test

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 4. Single-molecule co-localization of UV-DDB with SMUG1 on abasic DNA tightropes. (A) Schematic of the DNA tightrope assay. Long DNA substrates with abasic sites every 2 kb were suspended between 5 m poly-L-lysine coated silica beads. Anti-His primary antibody was used to link the His-tagged SMUG1 to the 605 Qdot. Biotin conjugated anti-Flag primary antibody was used to link Flag-tagged UV-DDB to streptavidin-coated 705 Qdot. Uniquely labeled SMUG1 and UV-DDB were observed interacting on abasic DNA tightropes in real-time and their behavior and frequency of co-localization were recorded. (B) Venn diagram showing the number of proteins that co-localized (yellow) on abasic (THF) tightropes or were observed separately for 605 Qdot-labeled SMUG1 (green) with 705 Qdot-labeled UV-DDB (red) in the dual-color assay. (C) Stacked bar graph showing the fraction of motile (gray) versus stationary (white) and persistent (solid) versus dissociating (diagonal lines). Results obtained with individual and co-localized particles. (SMUG1; SMUG1 behavior in the presence of UV-DDB, Co-local; co-localized SMUG1/UV-DDB behavior, UV-DDB; UV-DDB behavior in the presence of SMUG1). Data is re-plotted as a sub-set of (B). (D) Image of co-localized (yellow) Qdot-labeled SMUG1 (green) and UV-DDB (red) on abasic (THF) tightrope suspended between beads. The scale bar represents 2.5 m. Arrow points to co-localized particles. (E) Kymograph of co-localized SMUG1 and UV-DDB. Top, SMUG1 (green); middle, UV-DDB (red); bottom, merged (yellow). Horizontal and vertical scale bars represent 50 s and 2 kb, respectively.

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques: Labeling

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 5. DDB2 and SMUG1 co-localize in cells after treatment with 5-hmdU. (A) Immunofluorescence images demonstrating co-recruitment of DDB2- mCherry and SMUG1-GFP after labeling with 2.5 M EdU and 15-minute treatment with 5.0 M 5-hmdU. White arrow pointing towards co-localized foci. Scale bar 5 m. (B) Bar graph representing quantification of A (**** P < 0.0001; unpaired t-test). (C) Proximity ligation assay demonstrating co- recruitment of DDB2-mCherry and SMUG1-GFP after 15-min treatment with 5.0 M 5-hmdU. White arrows point towards nuclear PLA foci; yellow arrow is cytoplasmic PLA foci. Scale bar 5 m. (D) Bar graph representing quantification of (C) (**** P < 0.0001; unpaired t-test). (E) Quantitation of the kinetics of DDB2-mCherry and SMUG1-GFP recruitment after a 15-min treatment with 5-hmdU over the course of 4 h (*** P < 0.001, **** P < 0.0001; one way-ANOVA). N = 100–250 cells scored per time point.

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques: Immunofluorescence, Labeling, Proximity Ligation Assay, Quantitation Assay

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 6. Active repair of 5-hmdU results in cell toxicity driven by PARP1 activation. (A) Cell survival curves of U2OS cells transfected with either scrambled, SMUG1, DDB2, SMUG1/DDB2 targeting siRNA and treated with a range of concentrations of 5-hmdU, continuously for 8 days. Data represent mean ± SEM from three independent experiments, each performed in triplicate. (**p < 0.01; **** p < 0.0001; by two-way ANOVA). (B) Western blot analysis to confirm knockdown of SMUG1 and DDB2 after 8-day cell survival study, actin was used as loading control. (C) Immunofluorescence images demonstrating PAR expression in U2OS cells (WT, and after siRNA knockdown of SMUG1 or DDB2 treated with 5.0 M 5-hmdU and 20 M PARGi for 15 min, untreated controls received equal concentration DMSO. Images are representative of three biological experiments. Scale bar 5 m (D) Bar graph representing quantification of (C) (*** P < 0.001, ****P < 0.0001; one-way ANOVA).

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques: Activation Assay, Transfection, Western Blot, Knockdown, Control, Immunofluorescence, Expressing, Concentration Assay

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 7. Cellular bioenergetics during active 5-hmdU repair. (A) Basal oxygen consumption rate of U2OS WT, SMUG1-KD, DDB2-KD, SMUG1/DDB2-KD cells treated with 5-hmdU and PARGi. Untreated cells received DMSO control. (B) Basal extracellular acidification rate of U2OS WT, SMUG1-KD, DDB2-KD, SMUG1/DDB2-KD cells treated with 5-hmdU and PARGi. Untreated cells received DMSO control. Data represent mean ± SEM from 3-9 independent experiments performed with 6 repeats/experiment for 3 independent readings over time. (* P < 0.05, ** P < 0.01, **** P < 0.0001; one-way ANOVA).

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques: Control

Journal: Nucleic acids research

Article Title: UV-DDB stimulates the activity of SMUG1 during base excision repair of 5-hydroxymethyl-2'-deoxyuridine moieties.

doi: 10.1093/nar/gkad206

Figure Lengend Snippet: Figure 8. Working model for UV-DDB’s role in SMUG1 mediated removal of 5-hmdU during BER pathway. Schematic representation of the proposed BER pathway including UV-DDB. Red arrows indicate roles of UV-DDB in the pathway. Dashed arrows represent protein dissociation. UV-DDB appears to be rapidly recruited to damaged sites in chromatin and helps facilitate the processing of 5-hmdU by SMUG1. Biochemical and single molecule data suggest that UV-DDB transiently associates with SMUG1 at abasic sites to increase its turnover and stimulate BER, and cellular data supports transient interactions between UV-DDB and SMUG1 (see text for description).

Article Snippet: To obtain transient expression of SMUG1 and DDB2, U2OS cells were transfected with 500 ng each of SMUG1-GFP (Origene Cat# RG212141) and DDB2-mCherry (Gene Universal) using the lipofectamine 2000 reagent and protocol (Thermo Fisher Cat# 11668019).

Techniques:

Journal: Breast cancer research and treatment

Article Title: Single-strand selective monofunctional uracil-DNA glycosylase (SMUG1) deficiency is linked to aggressive breast cancer and predicts response to adjuvant therapy.

doi: 10.1007/s10549-013-2769-6

Figure Lengend Snippet: Fig. 1 SMUG1 mRNA expression in breast cancer. a Kaplan–Meier curves showing breast cancer-specific survival (BCSS). b Disease-free survival (DFS) in the training set

Article Snippet: As well as cell extracts from the cell lines, recombinant GST-tagged SMUG1 protein (Novus biological, USA) was simultaneously used as a positive control and to investigate the specificity of anti-SMUG1 antibody.

Techniques: Expressing