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Cell Signaling Technology Inc primary antibody against full length parp1
Primary Antibody Against Full Length Parp1, supplied by Cell Signaling Technology Inc, used in various techniques. Bioz Stars score: 98/100, based on 15003 PubMed citations. ZERO BIAS - scores, article reviews, protocol conditions and more
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MCELNs and CELNs elicited apoptosis in TNBC cells. (A)–(B) Flow cytometry detection of apoptosis in MDA-MB-231 cells treated with MCELNs and CELNs for 24 h. (C)–(D) Antagonism of MPA on cell apoptosis induced by MCELNs. (E)–(L) Western blot measured the expression levels of full length PARP1, cleaved PARP1, and cleaved caspase 3 following the treatment of MDA-MB-231 cells with MCELNs for 24 h ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Journal: Materials Today Bio

Article Title: Macrophage membrane coating enhances the therapeutic effects of Houttuynia cordata exosome-like nanovesicles against triple-negative breast cancer

doi: 10.1016/j.mtbio.2025.102604

Figure Lengend Snippet: MCELNs and CELNs elicited apoptosis in TNBC cells. (A)–(B) Flow cytometry detection of apoptosis in MDA-MB-231 cells treated with MCELNs and CELNs for 24 h. (C)–(D) Antagonism of MPA on cell apoptosis induced by MCELNs. (E)–(L) Western blot measured the expression levels of full length PARP1, cleaved PARP1, and cleaved caspase 3 following the treatment of MDA-MB-231 cells with MCELNs for 24 h ∗∗ p < 0.01, ∗∗∗ p < 0.001.

Article Snippet: The main materials used in this study included P-CHK1, P-ATR, CDK4, CDC25C, P21, γ-H2AX (Abclone), cleaved PARP1, full length PARP1, cleaved caspase 3, BSA (BioFroxx), BCA Protein Concentration Assay Kit (Biyotime Shanghai), SDS-PAGE protein loading buffer (BOSTER, Wuhan), Three-color prestained protein standards (Abclone), Cell Cycle Assay Kit (RedFluorescence) (Biyotime Shanghai), enhanced RIPA lysate (BOSTER, Wuhan), RPMI 1640 medium(Gibco, USA), PKH67 (MedChemExpress, USA), PEG8000 (Chron Chemicals), 5-Fluorouracil (MedChemExpress, USA), and Camptothecin (MedChemExpress, USA).

Techniques: Flow Cytometry, Western Blot, Expressing

a Upper panel—293T cells expressing Polθ-FLAG-HA. Middle panel—Detection of Polθ-FLAG-HA in anti-HA Immunoprecipitates (IP) with anti-FLAG antibody. Lower panel—intensity fold change of PARP1 in anti-HA IP from 293T cells expressing Polθ-FLAG-HA assessed by LC-MS/MS analysis. b STING database prediction of PARP1 interactions. c Polθ –PARP1 interaction detected by anti-HA and anti-PARP1 IP probed with anti-FLAG (Polθ-FLAG-HA) and anti-PARP1. d , e Co-localization of Polθ-FLAG-HA and PARP1 in d 293T cells and e MDA-MB-436 cells overexpressing Polθ-FLAG-HA and exposed to irradiation and 1 µM olaparib using confocal microscopy. Dimentions:10 μm or 100 nm (magnified foci). Quantification of yellow foci in cells ( n = 30) stems from three independent repeats: upper panels—mean number ± SD and lower panels -% of Polθ-FLAG-HA + PARP1 foci/cell. **** p < 0.0001 using one-way ANOVA ( d ) and two-tailed Student’s t test ( e ). f PARP1 PARylates Polθ-pol and Polθ-hel in vitro. SDS gel showing increase in the molecular weight (MW) of Polθ-pol and Polθ-hel in vitro by PARP1 PARylation. g PARP1 self-PARylation in vitro occurs in the presence of HPF1 predominantly on serine residues. SDS gel showing size increase of PARP1 due to self-PARylation. NH 2 OH treatment shows that serine residues of PARP1 are mainly self-PARylated in presence of HPF1. h HPF1 does not promote PARP1-NAD PARylation of Polθ-pol and Polθ-hel. SDS gel showing HPF1 promotes PARP1 self-PARylation on serine residues in the presence of NH 2 OH which prevents PARylation on glutamate and aspartate residues f – h represent at least two experiments. * indicates shift in MW in f – h . i , j PARP1-mediated PARylation of recombinant purified Polθ fragments: ΔPolθGS-FLAG (polymerase-helicase domain fusion) protein ( i ), and Polθ-pol and Polθ-hel ( j ). Purified p53-GST serves as control substrate. Western blot detected PARylation of Polθ and GST-p53 proteins. k , l Western blot detection of PARylated Polθ in anti-HA IP from ( k ) 10 Gy irradiated (IR) 293T (−) and Polθ-FLAG-HA transfected 293T cells treated or not with 1 µM olaparib (Ola), and ( l ) 293T cel l s (−) and these overexpressing PARP1 and/or Polθ-FLAG-HA. The panel represents three independent experiments. m Western blot detection of PARylated Polθ-FLAG-HA in anti-HA IPs from IR (+) or not (−) MDA-MB-436 cells and these expressing Polθ-FLAG-HA. Bottom panel: mean ± SD of the quantitation of PARylation from three independent biological replicates. *** p = 0.000185 using one-way ANOVA. n Co-localization of Polθ-FLAG-HA, PARP1, and/or PAR in MDA-MB-436 cells expressing Polθ-FLAG-HA and exposed to IR followed by 20 min incubation. Dashed line shows nuclear border. Scale bar: 10 μm or 100 nm (magnified foci). Co-localization experiments were repeated three times with n = 30 cells analyzed, and representative confocal microscopy images shown. Mean number ± SD of the indicated foci formation is shown below; **** p < 0.0001 using two-tailed Student’s t test. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

doi: 10.1038/s41467-024-50158-7

Figure Lengend Snippet: a Upper panel—293T cells expressing Polθ-FLAG-HA. Middle panel—Detection of Polθ-FLAG-HA in anti-HA Immunoprecipitates (IP) with anti-FLAG antibody. Lower panel—intensity fold change of PARP1 in anti-HA IP from 293T cells expressing Polθ-FLAG-HA assessed by LC-MS/MS analysis. b STING database prediction of PARP1 interactions. c Polθ –PARP1 interaction detected by anti-HA and anti-PARP1 IP probed with anti-FLAG (Polθ-FLAG-HA) and anti-PARP1. d , e Co-localization of Polθ-FLAG-HA and PARP1 in d 293T cells and e MDA-MB-436 cells overexpressing Polθ-FLAG-HA and exposed to irradiation and 1 µM olaparib using confocal microscopy. Dimentions:10 μm or 100 nm (magnified foci). Quantification of yellow foci in cells ( n = 30) stems from three independent repeats: upper panels—mean number ± SD and lower panels -% of Polθ-FLAG-HA + PARP1 foci/cell. **** p < 0.0001 using one-way ANOVA ( d ) and two-tailed Student’s t test ( e ). f PARP1 PARylates Polθ-pol and Polθ-hel in vitro. SDS gel showing increase in the molecular weight (MW) of Polθ-pol and Polθ-hel in vitro by PARP1 PARylation. g PARP1 self-PARylation in vitro occurs in the presence of HPF1 predominantly on serine residues. SDS gel showing size increase of PARP1 due to self-PARylation. NH 2 OH treatment shows that serine residues of PARP1 are mainly self-PARylated in presence of HPF1. h HPF1 does not promote PARP1-NAD PARylation of Polθ-pol and Polθ-hel. SDS gel showing HPF1 promotes PARP1 self-PARylation on serine residues in the presence of NH 2 OH which prevents PARylation on glutamate and aspartate residues f – h represent at least two experiments. * indicates shift in MW in f – h . i , j PARP1-mediated PARylation of recombinant purified Polθ fragments: ΔPolθGS-FLAG (polymerase-helicase domain fusion) protein ( i ), and Polθ-pol and Polθ-hel ( j ). Purified p53-GST serves as control substrate. Western blot detected PARylation of Polθ and GST-p53 proteins. k , l Western blot detection of PARylated Polθ in anti-HA IP from ( k ) 10 Gy irradiated (IR) 293T (−) and Polθ-FLAG-HA transfected 293T cells treated or not with 1 µM olaparib (Ola), and ( l ) 293T cel l s (−) and these overexpressing PARP1 and/or Polθ-FLAG-HA. The panel represents three independent experiments. m Western blot detection of PARylated Polθ-FLAG-HA in anti-HA IPs from IR (+) or not (−) MDA-MB-436 cells and these expressing Polθ-FLAG-HA. Bottom panel: mean ± SD of the quantitation of PARylation from three independent biological replicates. *** p = 0.000185 using one-way ANOVA. n Co-localization of Polθ-FLAG-HA, PARP1, and/or PAR in MDA-MB-436 cells expressing Polθ-FLAG-HA and exposed to IR followed by 20 min incubation. Dashed line shows nuclear border. Scale bar: 10 μm or 100 nm (magnified foci). Co-localization experiments were repeated three times with n = 30 cells analyzed, and representative confocal microscopy images shown. Mean number ± SD of the indicated foci formation is shown below; **** p < 0.0001 using two-tailed Student’s t test. Source data are provided as a Source Data file.

Article Snippet: Briefly, pET28a-based plasmid expressing the N-terminal HIS-tagged full-length PARP1 (1–1016 aa, Addgene plasmid #169815, https://www.addgene.org/169815 : RRID:Addgene_169815) was transformed into BL21(DE3) cells (Invitrogen).

Techniques: Expressing, Liquid Chromatography with Mass Spectroscopy, Irradiation, Confocal Microscopy, Two Tailed Test, In Vitro, SDS-Gel, Molecular Weight, Recombinant, Purification, Control, Western Blot, Transfection, Quantitation Assay, Incubation

a EMSA showing that PARylated Polθ-hel is inhibited in ssDNA binding. Cy3-ssDNA probe and PARP1 activating substrate (PAS) are indicated. b Denaturing gel showing that the presence of PARP1 and NAD+ inhibit Polθ-hel ATPase activity (left).% inorganic phosphate (PI) formation indicated. pssDNA substrate indicated. Bar plot showing mean ± SD results from ATPase assay (right). **** p < 0.0001 and *** p < 0.001 using unpaired two-tailed t test, statistics from three biological replicates (1 vs 3 p = 0.001119, 2 vs 3 p = 0.000025, 3 vs 4 p = 0.000045). c EMSA showing that the presence of PARP1 and NAD+ inhibit PolθΔcen ssDNA binding. Cy3-ssDNA probe and PARP1 activating substrate (PAS) are indicated. d Schematic of in vitro TMEJ assay using pssDNA substrates containing 3′ terminal microhomology. e Denaturing gel showing XmaI cleavage of expected TMEJ product. Schematic of pssDNA substrate and TMEJ synapse with XmaI recognition site indicated in red (top). f Denaturing gel showing that the presence of PARP1 and NAD+ suppress Polθ-pol and PolθΔcen TMEJ activities. g Bar plots showing mean% TMEJ activity ± SD by Polθ-pol and PolθΔcen in the presence of the indicated factors. **** p < 0.0001 using unpaired two-tailed t -test, statistics from three biological replicates (1 vs 3 p = 0.00002, 2 vs 3 p = 0.00001, 3 vs 4 p = 0.000025, 5 vs 7 p = 0.000086, 6 vs 7 p = 0.000011, 7 vs 8 p = 0.000035). h Denaturing gel showing that the presence of PARP1 and NAD+ suppress FL-Polθ TMEJ activity. i Bar plots showing mean ± SD results from%TMEJ activity by FL-Polθ and in the presence of the indicated factors. **** p < 0.0001 using unpaired two-tailed t -test, statistics from three biological replicates (1 vs 3 p = 0.000048, 2 vs 3 p < 0.00001, 3 vs 4 p < 0.00001). j Denaturing gel showing that olaparib prevents suppression of Polθ-pol TMEJ in the presence of PARP1 and NAD+. Experiments a , c , e , j were repeated at least two times with similar results. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

doi: 10.1038/s41467-024-50158-7

Figure Lengend Snippet: a EMSA showing that PARylated Polθ-hel is inhibited in ssDNA binding. Cy3-ssDNA probe and PARP1 activating substrate (PAS) are indicated. b Denaturing gel showing that the presence of PARP1 and NAD+ inhibit Polθ-hel ATPase activity (left).% inorganic phosphate (PI) formation indicated. pssDNA substrate indicated. Bar plot showing mean ± SD results from ATPase assay (right). **** p < 0.0001 and *** p < 0.001 using unpaired two-tailed t test, statistics from three biological replicates (1 vs 3 p = 0.001119, 2 vs 3 p = 0.000025, 3 vs 4 p = 0.000045). c EMSA showing that the presence of PARP1 and NAD+ inhibit PolθΔcen ssDNA binding. Cy3-ssDNA probe and PARP1 activating substrate (PAS) are indicated. d Schematic of in vitro TMEJ assay using pssDNA substrates containing 3′ terminal microhomology. e Denaturing gel showing XmaI cleavage of expected TMEJ product. Schematic of pssDNA substrate and TMEJ synapse with XmaI recognition site indicated in red (top). f Denaturing gel showing that the presence of PARP1 and NAD+ suppress Polθ-pol and PolθΔcen TMEJ activities. g Bar plots showing mean% TMEJ activity ± SD by Polθ-pol and PolθΔcen in the presence of the indicated factors. **** p < 0.0001 using unpaired two-tailed t -test, statistics from three biological replicates (1 vs 3 p = 0.00002, 2 vs 3 p = 0.00001, 3 vs 4 p = 0.000025, 5 vs 7 p = 0.000086, 6 vs 7 p = 0.000011, 7 vs 8 p = 0.000035). h Denaturing gel showing that the presence of PARP1 and NAD+ suppress FL-Polθ TMEJ activity. i Bar plots showing mean ± SD results from%TMEJ activity by FL-Polθ and in the presence of the indicated factors. **** p < 0.0001 using unpaired two-tailed t -test, statistics from three biological replicates (1 vs 3 p = 0.000048, 2 vs 3 p < 0.00001, 3 vs 4 p < 0.00001). j Denaturing gel showing that olaparib prevents suppression of Polθ-pol TMEJ in the presence of PARP1 and NAD+. Experiments a , c , e , j were repeated at least two times with similar results. Source data are provided as a Source Data file.

Techniques: Binding Assay, Activity Assay, ATPase Assay, Two Tailed Test, In Vitro

a Denaturing gel showing a time course of Polθ-pol TMEJ in the presence and absence of PARP1, NAD+, and PARG (left). Scatter plot showing a time course of Polθ-pol TMEJ in the presence and absence of PARP1, NAD+, and PARG (right); data represent mean ± SD results from three biological replicates. b Denaturing gels showing that PARG prevents inhibition of PolθΔcen and FL-Polθ TMEJ by PARP1 + NAD+. c Denaturing gel showing that PARG prevents inhibition of Polθ-pol TMEJ by PARP1 + NAD+. d Left: denaturing gel showing that the presence of HPF1 does not affect PARP1-NAD suppression or PARG rescue of Polθ-pol TMEJ activity in vitro. Right: Bar plot showing% MMEJ activity by Polθ-pol (pol) and in the presence of the indicated factors. Data represent mean ± SD from three biological replicates; *** p < 0.001, ** p < 0.01, and * p < 0.05 using unpaired two-tailed t -test (2 vs 4 p = 0.0003, 4 vs 5 p = 0.0052, 4 vs 7 p = 0.402, 6 vs 7 p = 0.0017, 7 vs 8 p = 0.0159). Experiments 3b–c were repeated two or three times with similar results. e , f Western analysis of the expression of PARP1 and PARG ( e ) and TMEJ activity: mean ± SD ratio of GFP + /dsRED+ cells from three biological replicates ( f ) in U2OS-EJ2-GFP cells transduced with the indicated shRNAs; *** p < 0.001 ( p value 1 vs 2 p = 0.0006, 1 vs 3 p = 0.0005) using one-way ANOVA. g , h Western analysis of PARylation ( g ) and TMEJ activity assay: mean ± SD ratio of GFP + /dsRED+ cells from three biological replicates ( h ) in U2OS-EJ2-GFP cells treated with the indicated inhibitors; **** p < 0.0001 using one-way ANOVA ( p value 1 vs 2 p < 0.00001, 1 vs 3 p < 0.00001). i , j Western analysis of HPF1 and ( i ) and TMEJ activity: mean ± SD ratio of GFP + /dsRED+ cells from three biolog i cal replicates ( j ) in U2OS-EJ2-GFP cells transduced with the indicated siRNAs. k , l U2OS cells (C) and these expressing Polθ-FLAG-HA were analyzed 120 min post-10 Gy irradiation. Irradiated cells were treated with PARP1i or PARGi. k Left: Polθ-FLAG-HA and γH2AX foci formation and co-localization. Dimentions:10 μm or 100 nm (magnified foci). Right : mean ± SD of Polθ-FLAG-HA - γH2AX yellow foci co-localization in individual nuclei ( n = 50) from two independent repeats. **** p < 0.0001 using one-way ANOVA. ( p value 1 vs 2 p < 0.00001, 2 vs 3 p < 0.00001, 2 vs 4 p < 0.000032). l Left: western blot analysis of anti-γH2AX immunoprecipitates from chromatin extracts from three independent biological replicates. Right: mean ± SD of protein band intensity is shown. **** p < 0.0001, *** p < 0.001, and ** p < 0.01 using one-way ANOVA. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

doi: 10.1038/s41467-024-50158-7

Figure Lengend Snippet: a Denaturing gel showing a time course of Polθ-pol TMEJ in the presence and absence of PARP1, NAD+, and PARG (left). Scatter plot showing a time course of Polθ-pol TMEJ in the presence and absence of PARP1, NAD+, and PARG (right); data represent mean ± SD results from three biological replicates. b Denaturing gels showing that PARG prevents inhibition of PolθΔcen and FL-Polθ TMEJ by PARP1 + NAD+. c Denaturing gel showing that PARG prevents inhibition of Polθ-pol TMEJ by PARP1 + NAD+. d Left: denaturing gel showing that the presence of HPF1 does not affect PARP1-NAD suppression or PARG rescue of Polθ-pol TMEJ activity in vitro. Right: Bar plot showing% MMEJ activity by Polθ-pol (pol) and in the presence of the indicated factors. Data represent mean ± SD from three biological replicates; *** p < 0.001, ** p < 0.01, and * p < 0.05 using unpaired two-tailed t -test (2 vs 4 p = 0.0003, 4 vs 5 p = 0.0052, 4 vs 7 p = 0.402, 6 vs 7 p = 0.0017, 7 vs 8 p = 0.0159). Experiments 3b–c were repeated two or three times with similar results. e , f Western analysis of the expression of PARP1 and PARG ( e ) and TMEJ activity: mean ± SD ratio of GFP + /dsRED+ cells from three biological replicates ( f ) in U2OS-EJ2-GFP cells transduced with the indicated shRNAs; *** p < 0.001 ( p value 1 vs 2 p = 0.0006, 1 vs 3 p = 0.0005) using one-way ANOVA. g , h Western analysis of PARylation ( g ) and TMEJ activity assay: mean ± SD ratio of GFP + /dsRED+ cells from three biological replicates ( h ) in U2OS-EJ2-GFP cells treated with the indicated inhibitors; **** p < 0.0001 using one-way ANOVA ( p value 1 vs 2 p < 0.00001, 1 vs 3 p < 0.00001). i , j Western analysis of HPF1 and ( i ) and TMEJ activity: mean ± SD ratio of GFP + /dsRED+ cells from three biolog i cal replicates ( j ) in U2OS-EJ2-GFP cells transduced with the indicated siRNAs. k , l U2OS cells (C) and these expressing Polθ-FLAG-HA were analyzed 120 min post-10 Gy irradiation. Irradiated cells were treated with PARP1i or PARGi. k Left: Polθ-FLAG-HA and γH2AX foci formation and co-localization. Dimentions:10 μm or 100 nm (magnified foci). Right : mean ± SD of Polθ-FLAG-HA - γH2AX yellow foci co-localization in individual nuclei ( n = 50) from two independent repeats. **** p < 0.0001 using one-way ANOVA. ( p value 1 vs 2 p < 0.00001, 2 vs 3 p < 0.00001, 2 vs 4 p < 0.000032). l Left: western blot analysis of anti-γH2AX immunoprecipitates from chromatin extracts from three independent biological replicates. Right: mean ± SD of protein band intensity is shown. **** p < 0.0001, *** p < 0.001, and ** p < 0.01 using one-way ANOVA. Source data are provided as a Source Data file.

Techniques: Inhibition, Activity Assay, In Vitro, Two Tailed Test, Western Blot, Expressing, Transduction, Irradiation

U2OS cells were irradiated with 10 Gy at time 0 followed by incubation for 20, 60, and 120 min. a Left: panels show representative Polθ-FLAG-HA, PARP1, PARG, and PAR nuclei ( n = 45) foci formation after irradiation from two independent biological replicates. Dimentions:10 μm or 100 nm (for magnified foci). Right - Dot-plots illustrating the mean arbitrary unit intensity ± SD of the indicated foci in individual nuclei; **** p < 0.0001 using one-way ANOVA. b Left: Polθ-FLAG-HA, PARP1, PARG and PAR foci formation and co-localization in U2OS cells after 10 Gy irradiation. Dimentions:10 μm or 100 nm (for magnified foci). Right: mean ± SD of Polθ-FLAG - HA—PARP1, Polθ-FLAG-HA–PARG, and Polθ-FLAG-HA—PAR yellow foci co-localization in individual nuclei ( n = 50) from two independent biological replicates. **** p < 0.0001 using one-way ANOVA c Left: Western blot analysis of anti-HA immunoprecipitates obtained post-10 Gy irradiation from total cell extracts of U2OS-EJ2-GFP cells (−) and these expressing Polθ-FLAG-HA from three independent biological replicates. Right: mean ± SD of protein band intensity is shown; ** p < 0.01 and *** p < 0.001 using one-way ANOVA. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

doi: 10.1038/s41467-024-50158-7

Figure Lengend Snippet: U2OS cells were irradiated with 10 Gy at time 0 followed by incubation for 20, 60, and 120 min. a Left: panels show representative Polθ-FLAG-HA, PARP1, PARG, and PAR nuclei ( n = 45) foci formation after irradiation from two independent biological replicates. Dimentions:10 μm or 100 nm (for magnified foci). Right - Dot-plots illustrating the mean arbitrary unit intensity ± SD of the indicated foci in individual nuclei; **** p < 0.0001 using one-way ANOVA. b Left: Polθ-FLAG-HA, PARP1, PARG and PAR foci formation and co-localization in U2OS cells after 10 Gy irradiation. Dimentions:10 μm or 100 nm (for magnified foci). Right: mean ± SD of Polθ-FLAG - HA—PARP1, Polθ-FLAG-HA–PARG, and Polθ-FLAG-HA—PAR yellow foci co-localization in individual nuclei ( n = 50) from two independent biological replicates. **** p < 0.0001 using one-way ANOVA c Left: Western blot analysis of anti-HA immunoprecipitates obtained post-10 Gy irradiation from total cell extracts of U2OS-EJ2-GFP cells (−) and these expressing Polθ-FLAG-HA from three independent biological replicates. Right: mean ± SD of protein band intensity is shown; ** p < 0.01 and *** p < 0.001 using one-way ANOVA. Source data are provided as a Source Data file.

Techniques: Irradiation, Incubation, Western Blot, Expressing

a Western blot validates HPF1 knockout in U2OS HPF1 KO cells. b Left: western blot analysis of anti-HA and anti- γH2AX immunoprecipitates obtained post-10 Gy irradiation from chromatin extracts of U2OS WT (−) and HPF1 KO cells (−) and these expressing Polθ-FLAG-HA. Right: mean ± SD of protein bands intensity quantification from three independent biological replicates is shown; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA. c Left: western blot analysis of anti-γH2AX immunoprecipitates obtained post-10 Gy irradiation from chromatin extracts of U2OS WT cells (−) and these expressing Polθ-FLAG-HA. Right: mean ± SD of protein bands intensity quantification from three independent biological replicates is shown; * p < 0.05, ** p < 0.01, and *** p < 0.001 using one-way ANOVA. d Left: Post-10 Gy irradiation co-localization of γH2AX with Polθ-FLAG-HA in U2OS WT and HPF 1KO cells expressing Polθ-FLAG-HA. Dimentions:10 μm or 100 nm (for magnified foci). Right: quantification of mean number ± SD of γH2AX-Flag - Polθ yellow foci formation ( n = 50 cells) from two independent biological replicates is shown; **** p < 0.0001 using one-way ANOVA. e Left: Post-10 Gy irradiation co-localization of γH2AX with PARP1 and PARG in U2OS cells expressing Polθ-FLAG-HA. Dimentions:10 μm or 100 nm (for magnified foci). Right: Quantification of mean number ± SD of γH2AX–PARP1 and γH2AX–PARG yellow foci formation ( n = 50) from two independent biological replicates is shown; **** p < 0.0001 using one-way ANOVA. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

doi: 10.1038/s41467-024-50158-7

Figure Lengend Snippet: a Western blot validates HPF1 knockout in U2OS HPF1 KO cells. b Left: western blot analysis of anti-HA and anti- γH2AX immunoprecipitates obtained post-10 Gy irradiation from chromatin extracts of U2OS WT (−) and HPF1 KO cells (−) and these expressing Polθ-FLAG-HA. Right: mean ± SD of protein bands intensity quantification from three independent biological replicates is shown; * p < 0.05, ** p < 0.01, *** p < 0.001, and **** p < 0.0001 using one-way ANOVA. c Left: western blot analysis of anti-γH2AX immunoprecipitates obtained post-10 Gy irradiation from chromatin extracts of U2OS WT cells (−) and these expressing Polθ-FLAG-HA. Right: mean ± SD of protein bands intensity quantification from three independent biological replicates is shown; * p < 0.05, ** p < 0.01, and *** p < 0.001 using one-way ANOVA. d Left: Post-10 Gy irradiation co-localization of γH2AX with Polθ-FLAG-HA in U2OS WT and HPF 1KO cells expressing Polθ-FLAG-HA. Dimentions:10 μm or 100 nm (for magnified foci). Right: quantification of mean number ± SD of γH2AX-Flag - Polθ yellow foci formation ( n = 50 cells) from two independent biological replicates is shown; **** p < 0.0001 using one-way ANOVA. e Left: Post-10 Gy irradiation co-localization of γH2AX with PARP1 and PARG in U2OS cells expressing Polθ-FLAG-HA. Dimentions:10 μm or 100 nm (for magnified foci). Right: Quantification of mean number ± SD of γH2AX–PARP1 and γH2AX–PARG yellow foci formation ( n = 50) from two independent biological replicates is shown; **** p < 0.0001 using one-way ANOVA. Source data are provided as a Source Data file.

Techniques: Western Blot, Knock-Out, Irradiation, Expressing

a – c Confocal images showing that PARP1 + NAD+ promote biomolecular condensates containing Cy3-DNA and Red-Polθ-pol. Confocal microscopy images are shown of Red-Polθ-pol and Cy3-DNA under the indicated conditions following Cy3 and Cy5 fluorescence as indicated. 10 μm bars are indicated. d , e Plots showing circularity ( d ) and size ( e ) of particles formed under the indicated conditions. Red circles represent particles with circularity scores >0.9 ( d ). f Confocal images showing that Red-Polθ-pol colocalizes with Cy3-DNA in droplets formed by PARP1 + NAD + . Confocal microscopy images are shown of Red-Polθ-pol and Cy3-DNA under the indicated conditions – following Cy3 and Cy5 fluorescence as indicated. 10 μm bars are indicated. g Quantitation of the number of co-localization events between Red-Polθ-pol and Cy3-DNA under the indicated conditions. Co-localization events from 4 separate confocal microscopy images are quantitated. h Model of Polθ TMEJ regulation by PARP1 and PARG. Experiments in 6 a – c , f were performed at least twice. Source data are provided as a Source Data file.

Journal: Nature Communications

Article Title: PARG is essential for Polθ-mediated DNA end-joining by removing repressive poly-ADP-ribose marks

doi: 10.1038/s41467-024-50158-7

Figure Lengend Snippet: a – c Confocal images showing that PARP1 + NAD+ promote biomolecular condensates containing Cy3-DNA and Red-Polθ-pol. Confocal microscopy images are shown of Red-Polθ-pol and Cy3-DNA under the indicated conditions following Cy3 and Cy5 fluorescence as indicated. 10 μm bars are indicated. d , e Plots showing circularity ( d ) and size ( e ) of particles formed under the indicated conditions. Red circles represent particles with circularity scores >0.9 ( d ). f Confocal images showing that Red-Polθ-pol colocalizes with Cy3-DNA in droplets formed by PARP1 + NAD + . Confocal microscopy images are shown of Red-Polθ-pol and Cy3-DNA under the indicated conditions – following Cy3 and Cy5 fluorescence as indicated. 10 μm bars are indicated. g Quantitation of the number of co-localization events between Red-Polθ-pol and Cy3-DNA under the indicated conditions. Co-localization events from 4 separate confocal microscopy images are quantitated. h Model of Polθ TMEJ regulation by PARP1 and PARG. Experiments in 6 a – c , f were performed at least twice. Source data are provided as a Source Data file.

Techniques: Confocal Microscopy, Fluorescence, Quantitation Assay

Overexpression (OE) of HIF1A-AS1 affects the proliferation, apoptosis of the pancreatic cancer (PC) cells (PANC-1). (A) RT-qPCR measure the mRNA level of HIF1A-AS1 in pancreatic cells (PANC-1), Real-time PCR showed that the levels of HIF1A-AS1 was significantly increased in the cells from OE group compared with the empty vector-controlled normal control (NC) cells. (B, C) OE of HIF1A-AS1 reduces significantly the viability of PANC-1 cells at 24, 48, and 72 h. (D) OE of HIF1A-AS1 promotes significantly apoptosis of PANC-1 cells assessment of cellular apoptosis using Annexin V‑fluorescein isothiocyanate staining coupled with flow cytometry. (E) Total percentage of apoptotic PANC-1 cells in each group are summarized with data presented as the mean ± SD of three independent experiments. (F) Western blotting reveal the full length caspase-3, Bax, P53, and PARP1 protein expression in PANC-1 cells. *** P <0.001. We used IMAGE J to analyze the expression of the target protein in western blot results. The specific data are as follows: (a) The protein expression increased about 22% for OE-Bax VS NC-Bax. (b) The protein expression increased about 20% for OE-caspase-3 VS NC-caspase-3. (c) The protein expression increased about 10% for OE-P53 VS NC-P53. (d) The protein expression increased about 34% for OE- PARP1 VS NC- PARP1.

Journal: Annals of Medicine and Surgery

Article Title: Deciphering the molecular mechanism of long non-coding RNA HIF1A-AS1 regulating pancreatic cancer cells

doi: 10.1097/MS9.0000000000002097

Figure Lengend Snippet: Overexpression (OE) of HIF1A-AS1 affects the proliferation, apoptosis of the pancreatic cancer (PC) cells (PANC-1). (A) RT-qPCR measure the mRNA level of HIF1A-AS1 in pancreatic cells (PANC-1), Real-time PCR showed that the levels of HIF1A-AS1 was significantly increased in the cells from OE group compared with the empty vector-controlled normal control (NC) cells. (B, C) OE of HIF1A-AS1 reduces significantly the viability of PANC-1 cells at 24, 48, and 72 h. (D) OE of HIF1A-AS1 promotes significantly apoptosis of PANC-1 cells assessment of cellular apoptosis using Annexin V‑fluorescein isothiocyanate staining coupled with flow cytometry. (E) Total percentage of apoptotic PANC-1 cells in each group are summarized with data presented as the mean ± SD of three independent experiments. (F) Western blotting reveal the full length caspase-3, Bax, P53, and PARP1 protein expression in PANC-1 cells. *** P <0.001. We used IMAGE J to analyze the expression of the target protein in western blot results. The specific data are as follows: (a) The protein expression increased about 22% for OE-Bax VS NC-Bax. (b) The protein expression increased about 20% for OE-caspase-3 VS NC-caspase-3. (c) The protein expression increased about 10% for OE-P53 VS NC-P53. (d) The protein expression increased about 34% for OE- PARP1 VS NC- PARP1.

Article Snippet: Membranes were incubated at room temperature for 1 h in a 5% skim milk TBST blocking solution and agitated with specific primary antibodies anti-GAPDH (Beyotime), anti-Bax (Cell signaling technology), anti-P53 (Szybio), anti- the full-length Caspase-3 (Cell Signaling Technology), and anti- the full-length PARP1 (Cell Signaling Technology) at 4°C overnight.

Techniques: Over Expression, Quantitative RT-PCR, Real-time Polymerase Chain Reaction, Plasmid Preparation, Control, Staining, Flow Cytometry, Western Blot, Expressing

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