Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 interacts with RAD51 at sites of DSB repair. (A) HaCaT cells synchronized in S phase were treated with bleomycin (12.5 µg/ml) for 30 min and released in fresh medium for 90 min or were untreated (control) followed by immunostaining with antibodies against S100A11 (green) and γH2AX (red). Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. (B) S phase HaCaT cells that were analyzed by laser scanning microscopy for S100A11 (green) and RAD51 (red) at sites of DNA damage 2 hour after bleomycin treatment. Bar, 10 µm. An area marked by a rectangle (light blue) in the overlay image (merge) is shown enlarged below the respective image. The intensities of the immunofluorescences in one cell derived from the RAD51 signal (red) and the S100A11 signal (green) are shown in a linescan (left side of the enlarged overlay). (C) Quantification of the colocalization of S100A11 with γH2AX (white Whisker box), RAD51 (gray Whisker box) and γH2AX/RAD51 (black Whisker box) in HaCaT cells after DSB induction. Colocalization events were determined in nuclei of cells treated as described in A. Thirty nuclei from 2 independent experiments were analyzed. Data are displayed as mean values (±SD). (D) Co-immunoprecipitation (CoIP) experiments between S100A11 and RAD51. A specific anti-S100A11 antibody precipitated RAD51 from whole cell extracts of S phase HaCaT cells treated with bleomycin (12.5 µg/ml) (lane 4) or untreated S phase cells (lane 6). In a control, an unspecific antibody used in CoIP experiments was unable to precipitate RAD51 from the same extracts (lanes 3 and 5). (E) The authenticity of the RAD51/S100A11 interaction was confirmed by pull-down with overexpressed proteins. U2OS cells were transfected with a plasmid encoding FLAG-S100A11 alone or together with a His-RAD51 encoding plasmid followed by synchronization in S phase and induction of DSBs by bleomycin treatment. Pull-down (pd) was carried out from cell extracts of the transfected cells for His-tagged proteins binding to Talon resins, and immunoblotting was performed using the antibodies as indicated. As loading control (load), 5% of the cell extract was used.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Control, Immunostaining, Laser-Scanning Microscopy, Derivative Assay, Whisker Assay, Immunoprecipitation, Transfection, Plasmid Preparation, Binding Assay, Western Blot

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 is functionally required for complete DSB repair. (A) Down-regulation of S100A11 in HaCaT cells treated with specific siRNA against S100A11 (lane 2: S100A11 siRNA#6, lane 3: S100A11 siRNA#7) was confirmed by immunoblotting using a specific antibody against S100A11. As control, HaCaT cells transfected with nonspecific control (nsc) siRNA (lane1) were used. GAPDH served as loading control. (B) Experimental setup. HaCaT cells were transfected with the indicated siRNA and subsequently synchronized in S phase by double-thymidine block. DNA damage was induced by treatment with 12.5 µg/ml bleomycin for 30 min. Cells were harvested after different time points (2 and 8 h repair) for immunostaining against S100A11 and γH2AX. (C) γH2AX foci persist in damaged S100A11 knock-down cells. Control represents cells without bleomycin treatment. The number of γH2AX foci per cell is shown as Whisker graphs. Mean values represent 3 independent experiments with n > 30 cells analyzed for each condition. (D) Analysis of the percentage of individual cells possessing γH2AX foci. HaCaT cells were treated as described in B. Mean values represent analysis of n > 90 cells for each condition in 3 independent experiments. (E) RAD51 foci (white) persist upon DSB damage after S100A11 knock-down. HaCaT cells were treated as described in B and analyzed by immunostaining against RAD51 and S100A11. Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. (F) Quantification of percentage of individual cell showing RAD51 foci. Mean values represent analysis of n > 85 cells for each condition in 3 independent experiments. * P < 0.05, *** P < 0.001.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Western Blot, Control, Transfection, Blocking Assay, Immunostaining, Knockdown, Whisker Assay

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: S100A11 stimulates the strand exchange activity of RAD51. (A) left panel Scheme of the strand exchange reaction between circular ssDNA and linearized dsDNA. right panel Strand exchange by human RAD51 requires Ca2+. RAD51, derived from 2 distinct purification procedures, (lanes 2–3 and 5–6: 3 µM) was incubated with 24 µM circular ΦX174 ssDNA in strand exchange buffer containing 2 mM of either magnesium or calcium acetate for 15 min at 37°C followed by incubation with 2.4 µM RPA for 5 min and addition of 24 µM linearized ΦX174 dsDNA to initiate strand exchange reaction for 2 h at 37°C. Lane M: constructed joint molecule DNA product derived from ssDNA/dsDNA annealing used as marker (B) left panel S100A11 enhances RAD51-mediated strand exchange. RAD51 (lanes 4–6: 3 µM) alone or with S100A11 (lane 5: 2 µM, lane 6: 4 µM) was incubated as described in (A) in strand exchange buffer containing calcium acetate (2 mM). As negative control, S100A11 (lane 7: 4 µM) was incubated alone. The joint molecule product (jm) was visualized by GelStar staining. right panel Quantification of S100A11-stimulated joint molecule formation by RAD51. Average values of 3 independent experiments are shown with standard derivation. (C) Dialysis of S100A11 abrogated the stimulating effect of S100A11 on RAD51 activity. RAD51 (lanes 4–8 and 10) together with undialyzed S100A11 (lane 5), S100A11 dialyzed in EGTA containing buffer (lanes 7–9), or S100A11 dialyzed in buffer without EGTA (lane 10), was incubated with 24 µM circular ΦX174 ssDNA in strand exchange buffer containing 2 mM of either magnesium or calcium acetate for 15 min at 37°C followed by incubation with 2.4 µM RPA for 5 min and addition of 24 µM linearized ΦX174 dsDNA to initiate strand exchange reaction for 2 h at 37°C. *P < 0.05.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Activity Assay, Derivative Assay, Purification, Incubation, Construct, Marker, Negative Control, Staining

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: A S100A11 mutant without Ca2+-binding impairs DSB repair. (A) left panel Immunostaining of U2OS cells for γH2AX in cells expressing recombinant S100A11. Cells expressing S100A11ΔCa display significantly increased γH2AX levels 8 h after DSB induction. Cells transfected with a plasmid encoding S100A11wt or S100A11ΔCa, respectively, were synchronized in S phase, followed by treatment with 12.5 µg/ml bleomycin for 30 min. Cells were harvested 8 h after induction of DNA damage and analyzed by immunostaining against γH2AX (white). Nuclear DNA was detected using DAPI (blue). Bar, 10 µm. right panel Quantification of the percentage of cells showing γH2AX foci after S100A11wt (n = 88) or S100A11ΔCa (n = 99) transfection. The results of 3 independent experiments are presented. Data are shown as the mean ±SD. (B) Recombinant S100A11ΔCa expression increases RAD51 foci persistence. left panel U2OS cells were treated as above and analyzed by immunostaining against RAD51 (white). Nuclear DNA was detected using DAPI (blue). Bar, 10 µM. right panel Quantification of percentage of individual cells showing RAD51 foci. Mean values represent analysis of cells expressing S100A11wt (n = 94) or S100A11ΔCa (n = 99) of 3 independent experiments. (C) Expression of S100A11wt (lane 2) or S100A11ΔCa (lane 3) in pcDNA4-transfected U2OS cells was confirmed by immunoblotting against S100A11. An empty plasmid was used for mock-transfection of U2OS cells (lane 1). GAPDH served as loading control. (D) S100A11ΔCa mutant failed to interact with RAD51. U2OS cells were transfected with a plasmid encoding FLAG-S100A11 (wild-type or ΔCa mutant, respectively) alone or together with a His-RAD51 encoding plasmid followed by synchronization in S phase and induction of DSBs by bleomycin treatment. Pull-down (pd) was carried out from cell extracts of the transfected cells using Talon resins to precipitate His-tagged proteins together with interacting partners. For analysis of the pulled-down interacting proteins, immunoblotting was performed using the antibodies as indicated. As loading control (load), 5% of the cell extract was used. * P < 0.05, ** P < 0.01, *** P < 0.001.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Mutagenesis, Binding Assay, Immunostaining, Expressing, Recombinant, Transfection, Plasmid Preparation, Western Blot, Control

Journal: Cell Cycle

Article Title: S100A11 plays a role in homologous recombination and genome maintenance by influencing the persistence of RAD51 in DNA repair foci

doi: 10.1080/15384101.2016.1220457

Figure Lengend Snippet: Downregulation of S100A11 results in restricted recombination capacity, chromosomal aberrations, and reduced cell viability. (A) S100A11 knock-down leads to a significant decrease in SCE. (top) Representative examples show multiple SCEs (left nsc siRNA, right S100A11 siRNA; white arrows) and (bottom) quantification of SCE per metaphase of 2 independent experiments. HaCaT cells with or without S100A11 siRNA were treated with bleomycin (12.5 µg/ml) for 30 min, allowed to recover for 2 h and transferred to medium containing 25 µM BrdU. After 4 h, 0.2 µg/ml Colcemid was added in fresh medium for 18 h to collect cells in metaphase. At least 40 metaphases per data point were analyzed. Error bars represent standard errors of the mean. (B-D) S100A11 knock-down leads to a significant increase of chromosomal abberations. HaCaT cells transfected with S100A11 siRNA or nsc siRNA were treated with 12.5 µg/ml bleomycin for 30 min. After further culture for 20 h, 0.1 µg/ml Colcemid was added for another 6 h to enrich cells in metaphase. The number of chromosome breaks (B) and complex chromosome aberrations (CCA) (C) are shown. Chromosome breaks and CCA were scored in n = 160 metaphase cells for each condition for 2 independent experiments. Error bars represent the standard errors of means. (D) Representative metaphase nuclei containing chromosomal aberrations as detected in S100A11-depleted HaCaT cells. Arrows (red) point to chromatid breaks; arrowheads (blue) point to radial figures. These aberrations are typical for DSBs occurred after DNA replication. (E) S100A11 knock-down leads to a significant loss of cell viability after bleomycin treatment. HaCaT cells transfected with specific S100A11 siRNA or control nsc siRNA for 72 h were treated with bleomycin (12.5 µg/ml) for 30 min. After this, medium was exchanged and the cells cultured for another 10 days. Then, the number of colonies formed was determined using Clono-Counter software in 3 independent experiments. HaCaT cells transfected with the control nsc siRNA without bleomycin treatment were used as control. * P < 0.05, ** P < 0.01, ***P < 0.001.

Article Snippet: Anti-S100A11 rabbit polyclonal antibody ( {"type":"entrez-nucleotide","attrs":{"text":"BC001410","term_id":"33876180"}} BC001410 ; Protein Tech Group) at 1:1,000, anti-S100A11 sheep polyclonal antibody (PAS9364; Randox Laboratories Inc.) at 1:2,000, anti-RAD51 rabbit polyclonal antibody (sc-8349; Santa Cruz) at 1:200, anti-RAD51 goat polyclonal antibody (sc-7410; Santa Cruz) at 1:200, anti-GAPDH monoclonal antibody (5174; Cell Signaling) at 1.1,000, normal mouse IgG (500-M00; Pepro Tech Inc.) at 1:1,000, and anti-FLAG mouse monoclonal antibody (F1804; Sigma) at 1:500 were used in coimmunoprecipitation experiments and immunoblotting.

Techniques: Knockdown, Transfection, Control, Cell Culture, Software

Journal:

Article Title: Identification of ARTS-1 as a novel TNFR1-binding protein that promotes TNFR1 ectodomain shedding

doi: 10.1172/JCI13847

Figure Lengend Snippet: Characterization of ARTS-1 as a type II integral membrane protein. (a) Specificity of anti–ARTS-1 serum. Immunoblots were performed on membrane and cytosolic fractions from NCI-H292 cells using anti–ARTS-1 immune or preimmune serum. Competitive inhibition experiments were conducted by preincubation of anti–ARTS-1 immune serum with either BSA or the peptide epitope against which the anti–ARTS-1 immune serum was raised. (b) ARTS-1 is a membrane-associated protein. Membrane (M) and cytosolic (CY) protein fractions of human bronchial epithelial cells (HBECs) obtained via bronchial brushings (left panel), human bronchial epithelial cell lines (NCI-H292, BEAS-2B, BET-1A, and A549) (center panel), and primary cultures of normal human bronchial epithelial cells (NHBEs), HUVECs, and fibroblasts (right panel) were separated by SDS-PAGE, transferred to nitrocellulose membranes, and reacted with anti–ARTS-1 immune serum. (c–f) Colocalization of membrane-associated ARTS-1 and TNFR1 in human bronchial epithelial cells. Confocal immunofluorescence laser microscopy was performed on nonfixed, nonpermeabilized frozen sections of normal human bronchi (c and d) and on nonfixed, nonpermeabilized cytospin preparations of normal human bronchial epithelial cells obtained via bronchial brushings (e and f) using a murine IgG2b isotype control and preimmune serum (c and e) and anti-TNFR1 and anti–ARTS-1 antibodies (d and f). An annotated differential interference contrast image is shown in the bottom left panels. Arrows denote the apical cell membrane. C, cilia; BM, basement membrane; SM, submucosa; N, nucleus; L, lateral cell membrane; B, basal cell membrane.

Article Snippet: The following antibodies were used: anti–ARTS-1 and preimmune rabbit serum (1:1,500 dilution), anti-TNFR1 extracellular domain (2 μg/ml murine monoclonal IgG 2b ; Santa Cruz Biotechnology Inc.), murine IgG 2b isotype control (2 μg/ml; R&D Systems Inc.), Alexa Fluor 488 F(ab′)2 fragment of goat anti-rabbit IgG, and Alexa Fluor 633 F(ab′)2 fragment of goat anti-mouse IgG (4 μg/ml; Molecular Probes Inc., Eugene, Oregon, USA).

Techniques: Membrane, Western Blot, Inhibition, SDS Page, Immunofluorescence, Microscopy