Journal: PLoS Pathogens

Article Title: Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase

doi: 10.1371/journal.ppat.1009841

Figure Lengend Snippet: (A) Levels of mtDNA present in the cytosol of MeV-infected MCF7 cells over time. Cytosolic mtDNA was quantitated via qPCR using a mitochondrial Dloop primer set ( D ), and represented as fold increase relative to mock-treated cells. (B) Immunoprecipitation followed by qPCR. Upper left: immunoblot of cell lysate transfected with empty plasmid or plasmid expressing HA-tagged cGAS. Lower left: the relative location of qPCR primer sets on mtDNA. Right: enrichment of DNA fragments using anti-HA antibody to coprecipitate DNA in mock- or MeV-infected cells, represented as fold increase relative to mock-treated cells. DNA fragments were amplified by qPCR using five primer pairs for mtDNA and three primer pairs for genomic DNA (gDNA). (C) MCF7 cells were transfected with siRNA for the negative control (NC), MAVS, cGAS, or both. Left: immunoblot of the cell lysate. Right: cells were infected with MeV or VSV, and the RNA collected at 24 h post-infection (hpi) was analyzed for IFN-β expression by RT-qPCR. (D) MCF7 cells were co-transfected with pISRE-Luc which is induced by type I IFN, and phRL-TK(int-) as an internal control, and then infected with MeV. Upper: Cells were harvested at the indicated time and the luciferase activities were measured. Lower: Cell lysates were subjected to western blotting to detect endogenous STING and phosphorylation of STING caused by cGAS activation. (E) qPCR analysis of the mtDNA content of cells cultured in ddC to generate mtDNA-depleted cells. Representative image of MCF7 cells stained with PicoGreen nucleic acid stain. Scale bar = 10 μm. (F) MCF7 cells treated with or without ddC were infected with MeV or VSV, and the RNA collected at 24 hpi was subjected to RT-qPCR to analyze IFN-β expression. (G) Left: cGAS expression levels in MCF7, H441, and 293SLAM cells were confirmed by western blotting. Right: 293SLAM cells treated with or without ddC were infected with MeV, and IFN-β mRNA levels were measured by RT-qPCR. (H) 293SLAM cells were transfected with empty plasmid or plasmid expressing HA-cGAS, and then infected with MeV or VSV. RNA was collected at 24 hpi and IFN-β levels were measured by RT-qPCR. Data are representative of three independent experiments. Data are the mean value ± SD ( n = 3). Statistical significance was determined using one-way analysis of variance (ANOVA) followed by Dunnett’s multiple comparison test (A, C, D) or unpaired Student’s t -test (B, E–H); * P < 0.05; ** P < 0.01; ns, not significant ( P > 0.05).

Article Snippet: 293SLAM cells (HEK293 cells stably expressing an MeV receptor SLAM) [ ], Vero cells (from ATCC), Vero-hSLAM cells (Vero cells stably expressing SLAM) and MCF7 cells (human breast cancer cells; from the Cell Resource Center for the Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Miyagi, Japan) were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 5% fetal calf serum (FCS).

Techniques: Infection, Immunoprecipitation, Western Blot, Transfection, Plasmid Preparation, Expressing, Amplification, Negative Control, Quantitative RT-PCR, Control, Luciferase, Phospho-proteomics, Activation Assay, Cell Culture, Staining, Comparison

Journal: PLoS Pathogens

Article Title: Downregulation of mitochondrial biogenesis by virus infection triggers antiviral responses by cyclic GMP-AMP synthase

doi: 10.1371/journal.ppat.1009841

Figure Lengend Snippet: (A-C) Vero-hSLAM cells were transfected with siRNA for the NC or Mfn1. (A) Cell lysates analyzed to western blotting. (B) Cells infected with rMV-EGFP. At 16 hpi, mitochondria were stained with MitoTracker. Scale bar = 10 μm. (C) Mitochondria morphology of at least 30 cells per condition and in three independent experiments were classified as normal, elongated, or fragmented mitochondrial network. (D) Mfn1 knockdown cells were infected with the mock control or MeV, and cytosolic mtDNA levels were measured by qPCR, as described above. (E) Upper: Vero-hSLAM cells were treated with 3 μg/ml ActD or irradiated with 60 mJ/cm 2 UV-C. Mitochondria and nuclei were stained 7 h later with MitoTracker and Hoechst, respectively. Lower: Cells were transfected with plasmid expressing HA-tagged Mfn1 and then stained with antibodies to COX IV for mitochondria and to the HA tag, and Hoechst. Scale bar = 10 μm. (F) Mitochondrial morphology of ~30 cells per condition and in two experiments were classified as normal, elongated, or fragmented mitochondrial network. (G) Cells were irradiated with UV, treated with ActD for 7 h, transfected with HA-Mfn1 plasmid, or infected with MeV for 24 h. Upper: cells were harvested and cytosolic mtDNA was measured by qPCR, as described above. Lower: total RNA was subjected to RT-qPCR to analyze IFN-β mRNA. (H) Immunoblot of MCF7 cells transfected with siRNA for the NC or PGC-1α. (I) MCF cells transfected with siRNA for the NC or PGC-1α, and cytosolic mtDNA was quantified by qPCR at 5 d post-transfection. Data are represented as the relative number of PGC-1α knockdown cells to that of NC-transfected cells. (J) MCF7 cells were treated with the mock control or ddC for 3 d and then transfected with siRNA for the NC or PGC-1α. After 4 d, RNA was harvested and the mRNA levels of IFN-β were measured by RT-qPCR. Data are representative of three independent experiments. Data are the mean value ± SD ( n = 3). Statistical significance was determined using an unpaired Student’s t -test; * P < 0.05; ** P < 0.01; ns, not significant ( P > 0.05).

Article Snippet: 293SLAM cells (HEK293 cells stably expressing an MeV receptor SLAM) [ ], Vero cells (from ATCC), Vero-hSLAM cells (Vero cells stably expressing SLAM) and MCF7 cells (human breast cancer cells; from the Cell Resource Center for the Biomedical Research Institute of Development, Aging and Cancer, Tohoku University, Miyagi, Japan) were maintained in Dulbecco’s modified essential medium (DMEM) supplemented with 5% fetal calf serum (FCS).

Techniques: Transfection, Western Blot, Infection, Staining, Knockdown, Control, Irradiation, Plasmid Preparation, Expressing, Quantitative RT-PCR

Journal: Journal of Biological Chemistry

Article Title: BRCA1-BARD1 Complexes Are Required for p53Ser-15 Phosphorylation and a G1/S Arrest following Ionizing Radiation-induced DNA Damage

doi: 10.1074/jbc.m405372200

Figure Lengend Snippet: FIG. 4. siRNA-mediated repression of BRCA1 disrupts the G1/S checkpoint. A, MCF-7 cells were transfected with GFP or BRCA1 siRNA. At 60 h post-transfection, cells were untreated or irradiated with 6 Gy IR or 50 J/m2 UV light and then incubated for 16 h before being pulsed for 1 h with 20 M BrdUrd. Cells were subsequently stained with anti-BrdUrd-FITC antibodies and propidium iodide and then analyzed by flow cytometry. The graph illustrates percent relative BrdUrd-positive cells in DNA damage-treated samples compared with untreated samples from two independent experiments. The graph dem- onstrates that GFP siRNA-transfected cells effectively arrest in G1 after IR or UV radiation; however, BRCA1-depleted cells cannot arrest in G1 following exposure to IR. B, fluorescence-activated cell sorter profiles of propidium iodide-stained cells of a representative experiment, de- scribed in A, are shown and indicate that BRCA1-depleted cells accu- mulate in G2/M following exposure to IR but not UV light, compared with control cells, which arrest in G1. C, MCF-7 cells were transfected with siRNAs targeting GFP and BRCA1. At 72 h post-transfection, cells were treated with the indicated DNA-damaging agents then incubated for 4 h before cellular extracts were prepared and immunoblotted for p21 levels using anti-p21 antibodies. Cellular extracts were also immu- noblotted for -tubulin, which served as a loading control.

Article Snippet: The following primary antibodies were used: ATMSer-1981 (Rockland Diagnostic), ATM (2C-1, Genetex), p21 (Oncogene Research), Chk1Ser-317 (7), Chk1 (Santa Cruz Biotechnology), Chk2Thr-68 (Cell Signaling), Chk2 (Santa Cruz), p53Ser-15 (Cell Signaling), p53 (Novo Castra), Nbs1 (Novus), c-junSer-63 (Santa Cruz), Cdc25A (Santa Cruz), BRCA1 (Ab-1; Oncogene Research), BRCA1Ser-1387, BRCA1Ser-1423, and BRCA1Ser-1524 (14), BARD1 (provided by Dr. Richard Baer), and -tubulin (T5192; Sigma).

Techniques: Transfection, Irradiation, Incubation, Staining, Flow Cytometry, Fluorescence, Control

Journal: Journal of Biological Chemistry

Article Title: BRCA1-BARD1 Complexes Are Required for p53Ser-15 Phosphorylation and a G1/S Arrest following Ionizing Radiation-induced DNA Damage

doi: 10.1074/jbc.m405372200

Figure Lengend Snippet: FIG. 5. A model illustrating the role of BRCA1-BARD1 dimers in the G1/S checkpoint. Stimulation of ATM activity by autophospho- rylation following IR-induced DNA damage results in phosphorylation of BRCA1 (cycle 1), which is bound to BARD1. This phosphorylation event allows BRCA1-BARD1 complexes to act as an adaptor for p53, enabling it to be targeted for phosphorylation by ATM at Ser-15 (cycle 2). Consequently, the transcriptional activity of p53 is enhanced to induce p21, causing a G1/S arrest. In contrast, BRCA1-BARD1 com- plexes are not required for a UV-induced G1/S arrest. Instead this pathway involves activation of ATR, which consequently phosphoryl- ates the kinases Chk1 and Chk2, resulting in their activation. The Cdc25A phosphatase is then targeted by these kinases for phosphoryl- ation and is subsequently degraded, preventing it from dephosphoryl- ating/activating CDK2, which causes a G1/S arrest.

Article Snippet: The following primary antibodies were used: ATMSer-1981 (Rockland Diagnostic), ATM (2C-1, Genetex), p21 (Oncogene Research), Chk1Ser-317 (7), Chk1 (Santa Cruz Biotechnology), Chk2Thr-68 (Cell Signaling), Chk2 (Santa Cruz), p53Ser-15 (Cell Signaling), p53 (Novo Castra), Nbs1 (Novus), c-junSer-63 (Santa Cruz), Cdc25A (Santa Cruz), BRCA1 (Ab-1; Oncogene Research), BRCA1Ser-1387, BRCA1Ser-1423, and BRCA1Ser-1524 (14), BARD1 (provided by Dr. Richard Baer), and -tubulin (T5192; Sigma).

Techniques: Activity Assay, Phospho-proteomics, Activation Assay

Journal: Journal of Neuroscience

Article Title: Analysis of the Ataxia Telangiectasia Mutated-Mediated DNA Damage Response in Murine Cerebellar Neurons

doi: 10.1523/jneurosci.2055-06.2006

Figure Lengend Snippet: Figure7. PhosphorylationofspecificATMsubstrates.A,p53activationafterDNAdamagein cultured granule cells. After DNA damage, p53 is activated and stabilized as a result of several posttranslational modifications. Western blot analysis of cultured granule neuronal proteins extracted30minafterNCStreatmentshowsthatp53isphosphorylatedheavilyatSer18onlyin Atm/cells.Thisphosphorylationlasts4handthendeclines(datanotshown).Westernblot analysis of cerebellar protein extracts 30 min after irradiation show that pS957 SMC1 (B) after DNA damage seen only in irradiated Atm/ cerebellum.

Article Snippet: Membranes were then probed with the following antibodies: mouse anti-tubulin monoclonal antibody (Sigma), rabbit anti-phosphoSer15 of p53 (Cell Signaling Technology), rabbit anti-p53 (DO1 plus 421), and rabbit anti-phospho-Ser957 of SMC1 (a member of the structural maintenance of chromosome family) (Novus Biologicals, Littleton, CO).

Techniques: Cell Culture, Western Blot, Irradiation

Journal: Molecular Medicine Reports

Article Title: Radiation-induced dysfunction of energy metabolism in the heart results in the fibrosis of cardiac tissues

doi: 10.3892/mmr.2021.12482

Figure Lengend Snippet: Irradiation induces alterations to the extracellular matrix and mitochondrial metabolism. (A) Immunohistochemistry staining analysis of Col1a1 and Col3a1 in the heart tissues. Magnification, ×400. (B) Western blotting and (C) semi-quantitative analysis of Col1a1, Col3a1, Vimentin and CTGF. (D) Western blotting and (E) semi-quantitative analysis of metabolism related genes Fasn and Slc25al. Relative protein levels of Col1a1, Col3a1, Vimentin, CTGF, Fasn and Slc25al were normalized to Tubulin. (F) Electron micrographs of the cardiac mitochondria from the mice in sham-irradiated and 5-month groups after 16 Gy radiation. Left magnification, ×3,000; Middle magnification, ×6,000; Right magnification, ×20,000. Green arrows show the myofilaments with fuzzy boundaries. Red arrows show swollen mitochondria with cavitation. Measurement of (G) ATP levels and (H) lactic acid concentration in the sham-irradiated and 5-month mice heart tissues. *P<0.05, **P<0.01, ***P<0.001; # P<0.05; n=3 per group. Col1a1, collagen type 1 α 1 chain; Col3a1, collagen type III α 1 chain; CTGF, CCCTC-binding factor; Fasn, fatty acid synthase; Slc25a1, solute carrier family 25 member 1.

Article Snippet: The primary antibodies used in the present study were: Anti-Col1a1 polyclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BA0325), anti-Col3a1 monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. M00788), anti-CCCTC-binding factor (CTGF) polyclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. PB0570), anti-vimentin (VIM) monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BM0135), anti-fatty acid synthase (Fasn) monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BM4865), anti-solute carrier family 25 member 1 (Slc25a1) polyclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. A05995-2) and anti-α-tubulin monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. M03989-2).

Techniques: Irradiation, Immunohistochemistry, Staining, Western Blot, Concentration Assay, Binding Assay

Journal: Molecular Medicine Reports

Article Title: Radiation-induced dysfunction of energy metabolism in the heart results in the fibrosis of cardiac tissues

doi: 10.3892/mmr.2021.12482

Figure Lengend Snippet: Upregulation of metabolism-related proteins in the mouse heart 5 months after exposure to ionizing radiation.

Article Snippet: The primary antibodies used in the present study were: Anti-Col1a1 polyclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BA0325), anti-Col3a1 monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. M00788), anti-CCCTC-binding factor (CTGF) polyclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. PB0570), anti-vimentin (VIM) monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BM0135), anti-fatty acid synthase (Fasn) monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. BM4865), anti-solute carrier family 25 member 1 (Slc25a1) polyclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. A05995-2) and anti-α-tubulin monoclonal antibody (1:1,000; Wuhan Boster Biological Technology, Ltd.; cat. no. M03989-2).

Techniques: