Journal: Nucleic Acids Research

Article Title: The chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage

doi: 10.1093/nar/gkaf1201

Figure Lengend Snippet: HAP1 cells lacking HELLS display hypersensitivity to MMS combined with Olaparib. (A) Representative image of cell survival assay of HAP1 and HAP1-HELLS KO cells treated with indicated concentrations of MMS and Olaparib for 3 days. The media was changed, and cells were grown for two additional days. Data represent at least four independent experiments. (B) Cell survival assay of HAP1 and HAP1-HELLS KO cells treated with indicated concentrations of MMS for 4 days. Data represent at least four independent experiments. (C) Proliferation curves of HAP1 and HELLS KO cells are presented as the percentage of confluence for cells treated for 1 h with 20 µM MMS, 1 µM Olaparib, or their combination. Data are a representation of at least two independent experiments. (D) Cell survival followed by PARP1 degradation was assessed by CCK8. Western blot of WCE in HAP1 and HELLS KO cells probed for PARP1 indicates PARP1 degradation by iRucaparib-AP6. HAP1 and HELLS KO cells were exposed to 4 μM iRucaparib-AP6 and indicated doses of MMS for 48 h to induce PARP1 degradation and DNA alkylation damage. Data are mean ± SEM. n = 3 independent biological replicates. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test. (E) Cell survival followed by PARP1 downregulation was assessed by MTT assay. HAP1 and HELLS KO cells silenced with 50 nM PARP1-specific siRNA were treated with the indicated doses of MMS for 48 h. Western blot of WCE in HAP1 and HELLS KO cells probed for PARP1 indicates PARP1 downregulation by siRNA. Data are mean ± SEM. n = 3 independent biological replicates. Statistical significance was determined by Student’s t -test with Welch modification, not assuming equal SD among different groups (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001).

Article Snippet: Western blots utilized the indicated antibodies: HELLS (Cell Signaling), Actin (SigmaAldrich), GAPDH (Cell Signaling), PARP1 (Cell Signaling), PARP2 (Santa Cruz Biotechnology), PARG (Cell signaling), γ-H2AX (Cell Signaling), Rad51 (EMD Millipore and Abclonal), pATM (Santa Cruz), MPG (Abcam), APEX (Abclonal Technology), XRCC1 (Santa Cruz Biotechnology and Active Motif), XRCC5 (Abclonal Technology), DNA LIG III (Santa Cruz Biotechnology), POLβ (Abcam), MacroH2A (Abcam), and PAR binding reagent (MilliporeSigma).

Techniques: Clonogenic Cell Survival Assay, Western Blot, MTT Assay, Modification

Journal: Nucleic Acids Research

Article Title: The chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage

doi: 10.1093/nar/gkaf1201

Figure Lengend Snippet: HELLS-deficient cells exhibit increased levels of PAR (Poly-ADP ribose). (A) Representative images (Bar = 100 µM) and (B) fluorescence intensity of PAR upon indicated treatments in HAP1 and HAP1-HELLS KO cells. Data represent the mean fluorescence intensity of 200 cells per sample of two independent biological experiments. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test ( **** P < 0.0001). (C–E) Western blots of WCE in HAP1 and HELLS KO probed for PARP1 and PAR, indicating PARP1-specific PARylation. PAR level in (C) haploid and diploid HAP1 and HELLS KO clones, (D) HAP1 and HELLS KO cells treated with indicated doses of Olaparib, and (E) siRNA-mediated downregulation of PARP1 in HAP1 and HELLS KO cells.

Article Snippet: Western blots utilized the indicated antibodies: HELLS (Cell Signaling), Actin (SigmaAldrich), GAPDH (Cell Signaling), PARP1 (Cell Signaling), PARP2 (Santa Cruz Biotechnology), PARG (Cell signaling), γ-H2AX (Cell Signaling), Rad51 (EMD Millipore and Abclonal), pATM (Santa Cruz), MPG (Abcam), APEX (Abclonal Technology), XRCC1 (Santa Cruz Biotechnology and Active Motif), XRCC5 (Abclonal Technology), DNA LIG III (Santa Cruz Biotechnology), POLβ (Abcam), MacroH2A (Abcam), and PAR binding reagent (MilliporeSigma).

Techniques: Fluorescence, Western Blot, Clone Assay

Journal: Nucleic Acids Research

Article Title: The chromatin regulator HELLS mediates SSB repair and responses to DNA alkylation damage

doi: 10.1093/nar/gkaf1201

Figure Lengend Snippet: HELLS interacts genetically with PARP1 and the HR pathway. (A) HELLS and PARP1 co-expression was assessed based on RNA-seq data from TCGA. (B) HELLS and PARP1 co-expression was analyzed based on proteomic data from the CPTAC web portals. For each cancer type, the extracted, normalized expression levels for two genes (HELLS and PARP1) were determined. The co-expression and statistical significance were determined using the Pearson correlation coefficient analysis. (C) Cell survival followed by RAD51 inhibition was assessed by CCK8. HAP1 and HELLS KO cells were exposed to the indicated doses of RAD51 inhibitor, B02, for 48 h. Western blot of WCE in HAP1 and HELLS KO cells probed for RAD51, indicating its basal expression level. (D) HAP1 and HELLS KO cells were dosed with indicated concentrations of RAD51i and Olaparib for 48 h, and cell survival was assessed by CCK8. (E&F) HAP and HeLa parental and HELLS KO cells were dosed with indicated doses of RAD51 inhibitor and MMS for 48 h, and cell survival was assessed by CCK8. All the cell survival data are mean ± SEM. n = 3 independent biological replicates. (G) DNA strand breaks quantified by alkaline comet assay. HAP1 and HELLS KO cells were pre-treated with 30 µM RAD51 inhibitor for 1 h, followed by 500 µM MMS for 1 h. Data are the average of comet tail moments of 100 cells per sample. n = 2 independent biological experiments. (H) Cell survival followed by BRCA2 downregulation was assessed by CCK8. HAP1 and HELLS KO cells were transfected with 1nM siRNA-BRCA2 and were dosed with the indicated doses of MMS for 48 h. Asterisk colors denote the following comparisons: red- HAP1 siBRCA2 to HELLS KO siBRCA2; black- HELLS KO siRNA control to HELLS KO siBRCA2; green- HAP1 siRNA control to HAP1 siBRCA2. Western blot of WCE in HAP1 and HELLS KO cells probed for BRCA2, indicating downregulation of BRCA2 by siRNA for 6 days. Statistical significance was determined by two-way ANOVA with Tukey’s multiple comparisons test (* P < 0.05, ** P < 0.01, *** P < 0.001, **** P < 0.0001). (I) Proposed model of HELLS-mediated repair of SSBs in response to DNA alkylation damage.

Article Snippet: Western blots utilized the indicated antibodies: HELLS (Cell Signaling), Actin (SigmaAldrich), GAPDH (Cell Signaling), PARP1 (Cell Signaling), PARP2 (Santa Cruz Biotechnology), PARG (Cell signaling), γ-H2AX (Cell Signaling), Rad51 (EMD Millipore and Abclonal), pATM (Santa Cruz), MPG (Abcam), APEX (Abclonal Technology), XRCC1 (Santa Cruz Biotechnology and Active Motif), XRCC5 (Abclonal Technology), DNA LIG III (Santa Cruz Biotechnology), POLβ (Abcam), MacroH2A (Abcam), and PAR binding reagent (MilliporeSigma).

Techniques: Expressing, RNA Sequencing, Inhibition, Western Blot, Alkaline Single Cell Gel Electrophoresis, Transfection, Control

Journal: Dose-Response

Article Title: Gentiana lutea Root Extract Attenuates Radiation-Induced Damage in Human PBMCs in vitro

doi: 10.1177/15593258261435484

Figure Lengend Snippet: Relative fold change of genes expression involved in DNA repair processes: Ogg1, Parp1 , and Xrcc1 in PBMCs pretreated with 0.25, 0.5, 1 and 2 mg/mL of Gentiana lutea root extract and exposed to 0.5 and 2 Gy irradiation dose

Article Snippet: Expression levels of the target genes were measured by quantitative Real-time PCR on Applied Biosystems Real-Time 7500 system (Applied Biosystems, Inc., Foster City, CA) using TaqMan® gene expression assays: Parp1 (Hs00242302_m1), Ogg1 (Hs00213454_m1), Xrcc1 (Hs00959834_m1).

Techniques: Expressing, Irradiation