Journal: EMBO Molecular Medicine

Article Title: Aurora-A expressing tumour cells are deficient for homology-directed DNA double strand-break repair and sensitive to PARP inhibition

doi: 10.1002/emmm.201000068

Figure Lengend Snippet: MCF10A cells were infected with a lentivirus construct driving the expression of GFP (empty vector) or both GFP and Aurora-A (Aurora-A) and mixed in equal proportion with non-infected cells to provide an internal control. The mixed cells were exposed to 5-Gy X-ray, followed by a 24 h (A) or 4, 16 or 32 h (B and C) recovery. A. Immunofluorescence images of MCF10A cells stained for RAD51 (grey) and γ-H2AX (red). Green fluorescence indicates GFP expression by infected cells. DNA was counterstained with DAPI (blue). Arrowheads indicate empty vector infected or Aurora-A overexpressing cells. B, C. Quantification of RAD51 (B) and γ-H2AX (C) foci-positive nuclei. Immunofluorescence images of MCF10A were acquired as in (A). The cells were pre-treated with the Aurora-A inhibitor MLN8054 (Aurora-A + MLN8054) or vehicle, 1 h before irradiation. Nuclei containing more than three foci were scored as positive. The percentage of RAD51 (B) or γ-H2AX (C) positive nuclei of non-infected (GFP negative) or infected (GFP positive) cells was plotted. Error bars represent SEM. Results are representative of at least two independent experiments.

Article Snippet: Cells grown on a glass coverslip were fixed in PBS–4% paraformaldehyde for 1 h, permeabilized in PBS–0.5% Triton X-100 for 15 min, blocked in PBS–2%FCS–1%BSA, before incubation with rabbit-anti-RAD51 (Santa Cruz) and mouse-anti-γ-H2AX (Upstate) antibodies.

Techniques: Infection, Construct, Expressing, Plasmid Preparation, Control, Immunofluorescence, Staining, Fluorescence, Irradiation

Journal: The FASEB Journal

Article Title: Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy‐related progeroid syndromes

doi: 10.1096/fj.201700014r

Figure Lengend Snippet: Figure 1. XPA replaces PCNA at stalled replication forks in HGPS cells. A) Colocalization of g-H2AX foci and the foci of PCNA in BJ (UV-treated) cells but not in HGPS and RD fibroblasts. B) PCNA formed nuclear foci in young HGPS cells, whereas XPA formed foci in late-passage HGPS cells. C) Colocalization of MCM7 foci and the foci of g-H2AX and XPA in late-passage HGPS cells. Antigens were detected with corresponding antibodies by immunofluorescence micros- copy. Uniform staining of the given protein throughout the nucleus indicates homogenous distribution of the protein without foci formation.

Article Snippet: The fixed cells were incubated with primary antibodies against g-H2AX (rabbit, Bethyl or mouse; Stressgen, San Diego, CA, USA), XPA [mouse (Thermo Fisher Scientific) or rabbit (Santa Cruz Biotechnology)], MCM7 (mouse; Santa Cruz Biotechnology) or PCNA (rabbit; Santa Cruz Biotechnology).

Techniques: Staining

Journal: The FASEB Journal

Article Title: Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy‐related progeroid syndromes

doi: 10.1096/fj.201700014r

Figure Lengend Snippet: Figure 2. XPA-associated DSBs form at replication forks in HGPS. A) HGPS cells treated with or without CPT were subjected to the modified ChIP assay with pulldown by the anti-g-H2AX antibody. The immunoprecipitated chromatin was analyzed for PCNA and MCM7 Co-IP by Western blot analysis. PCNA was largely absent at the replication forks with endogenous progerin-induced strand breaks, but was present in chromatin at CPT-induced strand breaks. MCM7 is a replication fork marker. B) HGPS cells were transfected with XPA siRNA, then mock or CPT treated, followed by analysis using modified ChIP assay with anti-g-H2AX antibody. The successful knockdown of XPA by siRNA significantly restores the binding of PCNA and Pold to the replication forks in the progeroid cells.

Article Snippet: The fixed cells were incubated with primary antibodies against g-H2AX (rabbit, Bethyl or mouse; Stressgen, San Diego, CA, USA), XPA [mouse (Thermo Fisher Scientific) or rabbit (Santa Cruz Biotechnology)], MCM7 (mouse; Santa Cruz Biotechnology) or PCNA (rabbit; Santa Cruz Biotechnology).

Techniques: Immunoprecipitation, Co-Immunoprecipitation Assay, Western Blot, Marker, Transfection, Knockdown, Binding Assay

Journal: The FASEB Journal

Article Title: Progerin sequestration of PCNA promotes replication fork collapse and mislocalization of XPA in laminopathy‐related progeroid syndromes

doi: 10.1096/fj.201700014r

Figure Lengend Snippet: Figure 3. PCNA is sequestered by progerin in HGPS cells. A) XPA nuclear focus formation in normal BJ cells after PCNA knockdown by PCNA-specific siRNA. Western blot analysis demonstrated the efficiency of PCNA knockdown. B) Formation of XPA foci and colocalization with g-H2AX in BJ cells with increasing passage number. One hundred cells were randomly chosen and counted. Cells that had at least 3 XPA foci were regarded as foci-positive cells. Statistical analysis of the data indicated that the changes were statistically significant between passage numbers 16 and 33 and between 16 and 44 (x2 = 79; P , 0.01). Nuclear XPA is significantly associated with the chromatin in later passage BJ cells. Progerin accumulation with increasing passage number in BJ cells was analyzed by Western blot analysis of the whole-cell extracts. C) PLA using PCNA-specific and either lamin A- or progerin-specific antibodies in old-passage HGPS cells. A significant majority of PCNA was associated with progerin, not lamin A. After primary antibody incubation, secondary antibodies conjugated to complimentary oligonucleotides that were bound, ligated, and amplified to generate a red signal indicating interaction of the respective protein pairs. Interaction-positive foci were quantified and plotted. D) HGPS cells were subjected to a Co-IP assay with pulldown by the anti-progerin antibody. The immunoprecipitated progerin was analyzed for PCNA association by Western blot analysis. More PCNA pulldowns with progerin in untreated old HGPS cells (P18) than in younger HGPS cells (P9). The bead control contains HGPS cell lysate without antibody.

Article Snippet: The fixed cells were incubated with primary antibodies against g-H2AX (rabbit, Bethyl or mouse; Stressgen, San Diego, CA, USA), XPA [mouse (Thermo Fisher Scientific) or rabbit (Santa Cruz Biotechnology)], MCM7 (mouse; Santa Cruz Biotechnology) or PCNA (rabbit; Santa Cruz Biotechnology).

Techniques: Knockdown, Western Blot, Incubation, Co-Immunoprecipitation Assay, Immunoprecipitation, Control

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Hydrogen peroxide and hypoxia resulted in DNA damage, G2/M arrest and senescence in HK-2 cells. (A) Representative western blots analysis of γH2A.X (ser139), and bar graphs showed the fold changes compared to control group at different time points. (B) Flow cytometry analysis of cell cycle. * P < 0.05, **** P < 0.0001, compared with control group. (C) SA-β-gal staining analysis at different time points. (D) EdU incorporation analysis at 48 h. (E) Representative SA-β-gal staining at 48 h, Scale bars, 50 μm. (F) Representative EdU incorporation images at 48 h showed decreased proliferation rate and larger cell size in H 2 O 2 and hypoxia groups, Scale bars, 50 μm. n = 3–5/group. Data are means ± SD. SA-β-gal, Senescence Associated β-Galactosidase; EdU, 5-Ethynyl-2′-deoxyuridine.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Western Blot, Control, Flow Cytometry, Staining

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) attenuated hydrogen peroxide and hypoxia induced injuries and senescence in HK-2 cells. (A) Cell viability percentage of HK-2 cells. * P < 0.05, ** P < 0.01, *** P < 0.001 compared with H 2 O 2 or hypoxia group. (B) Quantification of NAD + levels in HK-2 cells. (C,D) Flow cytometry analysis of γH2A.X (ser139) in H 2 O 2 group, and bar graph showed decreased DNA damage in NMN-treated group. (E) Representative SA-β-gal staining images, Scale bars, 50 μm. (F) Bar graphs showing significantly decreased percentage of SA-β-gal positive cells in NMN-treated groups at a dose of 1 mM. (G,H) Western blots analysis showed reduction of collagen IV in NMN-treated groups, compared with H 2 O 2 or hypoxia group. n = 3–5/group. Data are means ± SD. NMN, Nicotinamide Mononucleotide.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Flow Cytometry, Staining, Western Blot

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) administration before ischemia and during acute phase attenuated tubular DNA damage and cellular injury in uIRI mice at day 3 after surgery. (A) Schematic protocol of the experiments. (B) Representative images of HE (hematoxylin-eosin) staining and histologic analysis of tubular damage. Scale bars, 100 μm. (C) Quantification of damaged tubules of ischemic kidneys in (B) . (D) Representative images of in situ TUNEL assay. Scale bars, 50 μm. (E) Quantification of apopotic tubular cells in ischemic kidneys in (D) . (F) Western blots analysis of γH2A.X (ser139) showed decreased DNA damage in ischemic kidneys of NMN-treated mice compared with ischemic kidneys of PBS-treated mice. (G) Representative images of co-immunostaining of anti-Ki-67 and anti-p-H3 in kidney tissues. Scale bars, 50 μm. (H) Bar graphs showed no changes in number of Ki-67 positives tubular cells in NMN-treated mice, but a markedly reduction of tubular cells in G2/M phase compared with PBS-treated mice. n = 6–10/group. Data are means ± SD.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Staining, In Situ, TUNEL Assay, Western Blot, Immunostaining

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) administration before ischemia and during acute phase attenuated tubular senescence in uIRI mice at day 21 after surgery. (A) Representative western blots analysis of γH2A.X (ser139). (B) Bar graph showed reduction of DNA damage in NMN-treated group. (C,D) SA-β-gal staining analysis showed decreased senescent tubular cells in NMN-treated uIRI mice. Scale bars, 50 μm. (E) Relative expression of IL-6, IL-8, and TGF-β1 mRNAs after normalization with β-actin. n = 6–7/group. Data are means ± SD.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Western Blot, Staining, Expressing

Journal: Frontiers in Physiology

Article Title: Nicotinamide Mononucleotide Attenuates Renal Interstitial Fibrosis After AKI by Suppressing Tubular DNA Damage and Senescence

doi: 10.3389/fphys.2021.649547

Figure Lengend Snippet: Nicotinamide mononucleotide (NMN) administration at recovery phase attenuated uIRI induced DNA damage, inflammation and fibrosis at day 21 after surgery. (A) Schematic protocol of the experiments. (B,C) Representative western blots analysis of γH2A.X (ser139), and bar graph showed reduction of DNA damage in NMN-treated group. (D) Representative images of SA-β-gal staining in both groups. Scale bars, 50 μm. (E) Quantification of expression of SA-β-gal in (D) . (F) Relative expression of IL-6, IL-8, and TGF-β1 mRNA after normalization with β-actin. (G,H) Representative sirius red staining images and semiquantitative analysis of the percentage of positive area. Scale bars, 100 μm. (I,J) Representative western blots and semiquantitative analysis of Collagen IV (Col IV), n = 6/group. Data are means ± SD.

Article Snippet: For analysis of DNA damage, HK-2 cells were incubated with a γH2A.X (ser139) antibody (CST, #9719) according to the manufacturer’s protocol.

Techniques: Western Blot, Staining, Expressing

Journal: International Journal of Oncology

Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

doi: 10.3892/ijo.2018.4553

Figure Lengend Snippet: DNA damage detection in TC-1 and B16 tumour cell lines. To detect DNA damage, control, DTX- or IFNγ + TNFα-treated (A) TC-1 and (B) B16 cells were stained with phosphoSer139 H2A histone family, member X antibody and mounted with Mowiol containing 4’,6-diamidine-2-phenylindole. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with DTX or IFNγ + TNFα was quantified. Data are presented as the mean ± standard deviation. * P<0.05, ** P<0.01 and *** P<0.001 vs. CTRL. A total of 100 cells were analysed in each experimental group. CTRL, control cells; DTX, docetaxel; IFNγ, interferon γ; TNFα, tumour necrosis factor α; B16, B16F10 cell line.

Article Snippet: The antibodies used were as follows: Phospho-Ser139 of histone H2A histone family, member X (γH2AX) rabbit monoclonal antibody (cat no. 9718; Cell Signaling Technology, Inc.) and goat anti-rabbit immunoglobulin G antibody conjugated with Alexa 488 (cat no. A11034; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Control, Staining, Standard Deviation

Journal: International Journal of Oncology

Article Title: Distinct phenotypes and ‘bystander’ effects of senescent tumour cells induced by docetaxel or immunomodulatory cytokines

doi: 10.3892/ijo.2018.4553

Figure Lengend Snippet: DNA damage response in ‘bystander’ cells treated with conditioned medium from senescent TC-1 and B16 cell lines. Immunofluorescence detection of phosphoSer139 H2A histone family, member X in (A) TC-1 and (B) B16 cells treated with relevant SM and TM medium for 4 days. Scale bar, 20 µ m. Percentage of cells with 1, 2, 3 or more micronuclei in (C) TC-1 and (D) B16 cells treated with SM or TM was quantified. Data are presented as the mean ± standard deviation. ** P<0.01 and *** P<0.001 vs. CTRL. A total of 100 cells were analysed in each experimental group. CTRL, control cells; SM, senescence medium; TM, tumour medium; B16, B16F10 cell line.

Article Snippet: The antibodies used were as follows: Phospho-Ser139 of histone H2A histone family, member X (γH2AX) rabbit monoclonal antibody (cat no. 9718; Cell Signaling Technology, Inc.) and goat anti-rabbit immunoglobulin G antibody conjugated with Alexa 488 (cat no. A11034; Invitrogen; Thermo Fisher Scientific, Inc.).

Techniques: Immunofluorescence, Standard Deviation, Control