Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Control lanes from a 384 well: 16 negative control wells contain stoichiometric RAD52- Cy3-dT 30 -Cy5 complexes (red filled circles), while 16 positive control wells contain a stoichiometric RAD52- Cy3-dT 30 -Cy5 complex challenged with unlabeled polydT100 (blue filled circles). Red and blue lines with error bars at the ends indicate the average and the standard deviation for the negative and positive controls, respectively. Z’ factor of 0.94 was calculated for these control lanes, indicating excellent reliability of the assay. ( b ) A representative 384 well plate from the HTS screen highlighting ‘1’ , ‘5’ , ‘6’ , ‘7’ , and ‘15’ . Red and blue lines with error bars at the ends indicate the average and the standard deviation for the negative and positive controls, respectively. ( c ) Average of cherry-picked rescreening of compounds identified from screening all plates in the MicroSource SPECTRUM collection highlighting all 12 identified hits (green filled circles) along with a number of false positive compounds (blue filled circles) that either showed poor reproducibility in subsequent rescreening or a linear dependence of the signal on the compound concentration. Red and blue lines with error bars at the ends indicate the average and the standard deviation for the negative and positive controls, respectively. DOI: http://dx.doi.org/10.7554/eLife.14740.003

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Negative Control, Positive Control, Standard Deviation, Concentration Assay

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: The twelve hits from the FRET-based HTS assay aimed at finding inhibitors of the RAD52-ssDNA interaction. DOI: http://dx.doi.org/10.7554/eLife.14740.004

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: HTS Assay, Binding Assay

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Aromatic region of the 1D 1 H NMR spectrum of compound ‘1’ alone (black) and the WaterLOGSY spectrum of 20 μM compound ‘1’ in the presence of 3.3 μM RAD52 (red). The nonexchangeable proton peaks are labeled using atom names as indicated on the structure of compound ‘1’. (b ) IC 50 values for inhibition of ssDNA binding and wrapping were determined using FRET-based assays that follow the change in geometry of a Cy3-dT30-Cy5 substrate (black circles). The computed IC 50 value is shown below the curve. Titration of the RAD52-dsDNA with ‘1’ (grey boxes) shows no perturbation of the dsDNA binding. ( c ) IC 50 values for inhibition of RAD52-mediated ssDNA annealing were determined by fitting the dependence of the extent of oligonucleotide-based annealing reaction carried in the presence of increasing concentration of ‘1’ . ( d ) Aromatic region of the 1D 1 H NMR spectrum of compound ‘1’ alone (black) and the WaterLOGSY spectrum of 20 μM compound ‘1’ in the presence of 3.3 μM RPA (red). ( e ) Titration of the RAD52-RPA- Cy3-dT 30 -Cy5 complex with ‘1’ (black circles). The computed IC 50 value is shown below the curve. Green squares show titration of the RPA- Cy3-dT 30 -Cy5 complex with ‘ 1 ’. ( f ) IC 50 values for inhibition of RAD52-mediated annealing of the RPA-coated ssDNA were determined by fitting the dependence of the extent of the annealing reaction carried out in the presence of increasing concentration of ‘ 1 ’. DOI: http://dx.doi.org/10.7554/eLife.14740.005

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Labeling, Inhibition, Binding Assay, Titration, Concentration Assay

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Aromatic region of the 1D 1 H NMR spectrum of compound ‘6’ alone (black) and the WaterLOGSY spectrum of 40 μM compound ‘6’ in the presence of 3.3 μM RAD52 (red). The nonexchangeable proton peaks are labeled using atom names as indicated on the structure of compound ‘6’ . ( b ) IC 50 values for inhibition of ssDNA binding and wrapping were determined using FRET-based assays that follow the change in geometry of a Cy3-dT30-Cy5 substrate (black circles). The computed IC 50 value is shown above the curve. Titration of the RAD52-dsDNA with ‘6’ (grey boxes) shows that this inhibitor also perturbs the RAD52-dsDNA interaction. ( c ) IC 50 values for inhibition of RAD52-mediated ssDNA annealing were determined by fitting the dependence of the extent of oligonucleotide-based annealing reaction carried in the presence of increasing concentration of ‘6’ . ( d ) Aromatic region of the 1D 1 H NMR spectrum of compound ‘6’ alone (black) and the WaterLOGSY spectrum of 40 μM compound ‘6’ in the presence of 3.3 μM RPA (red). ( e ) Titration of the RAD52-RPA- Cy3-dT 30 -Cy5 complex with ‘6’ (black circles). The computed IC 50 value is shown below the curve. Green squares show titration of the RPA- Cy3-dT 30 -Cy5 complex with ‘ 6 ’. ( f ) IC 50 values for inhibition of RAD52-mediated annealing of the RPA-coated ssDNA were determined by fitting the dependence of the extent of the annealing reaction carried out in the presence of increasing concentration of ‘ 6 ’. DOI: http://dx.doi.org/10.7554/eLife.14740.010

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Labeling, Inhibition, Binding Assay, Titration, Concentration Assay

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Schematic of the FRET based annealing reaction. In two half-reactions, stoichiometric amounts of RAD52 were incubated with Target28Cy3 and Probe28Cy5 oligonucleotides, respectively. Upon mixing of the two half-reactions, RAD52 facilitated annealing of the two complementary oligonucleotides, which can be observed as an increase in FRET between Cy3 and Cy5 dyes. ( b ) Annealing reactions performed in the absence (blue) or presence of increasing concentrations of ‘ 1 ’. The average of three or more independent annealing reactions is shown for each curve. Grey continuous lines show fits to double exponentials. Light green circles correspond to the DNA only reaction; dark green circles represent the annealing reaction containing the DNA and 100 µM ‘ 1 ’. DOI: http://dx.doi.org/10.7554/eLife.14740.007

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Incubation

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) FRET measurements were performed by titrating RAD52 protein into a solution containing 1 nM Cy3-dT 30 -Cy5 ssDNA. The Cy3 fluorescence was excited directly and the emissions of Cy3 and Cy5 dyes were measured, and the respective FRET values calculated as described in the Materials and methods. The highest separation in the FRET signal of unbound ssDNA and the RAD52-ssDNA complex was achieved around 8 nM RAD52. At this concentration, one RAD52 heptameric ring binds and wraps the ssDNA oligonucleotide bringing the Cy3 and Cy5 dyes close to one another. The details and control experiments for these measurements can be found in ( ; ). Each data point represents the average and standard deviation for at least three independent titrations. ( b ) The titrations were performed similarly to those shown in a , except 1 nM RPA was added to a solution containing 1 nM Cy3-dT 30 -Cy5 ssDNA prior to titration of RAD52. Under our experimental conditions, RPA forms stoichiometric complexes with the 30-mer DNA oligonucleotide with 1 RPA coating 1 molcule of ssDNA. The data points and error bars represent averages and standard deviation for three or more independent titrations. ( c ) The titrations were performed similarly to those shown in a , except 1 nM dsDNA (Cy3-Oligo28-Cy5 annealed to Oligo28-REV) was used as a substrate. Stoichiometric complexes are achieved at 10 nM RAD52. The data points and error bars represent averages and standard deviation for three or more independent titrations. DOI: http://dx.doi.org/10.7554/eLife.14740.006

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Fluorescence, Concentration Assay, Standard Deviation, Titration

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: A possible effect of the identified compounds on the oligomeric state of RAD52 protein was probed in the dynamic light scattering experiments, which measured the average hydrodynamic radius of RAD52 (15.8 μM) alone or in the presence of equimolar concentrations of each compound. Measurements were recorded at 25°C in buffer containing 50 mM Tris-HCl pH7.5, 200 mM KCl, 1 mM DTT, and 0.5 mM EDTA. A total of 10 measurements with 10 accumulations were collected, had monomodal distributions, and sum of squares (SOS) values of ≤ 0.65. Measurements were recorded with a DynaPro NanoStar (Wyatt Tech. Corp.) and the hydrodynamic radius were calculated by the DYNAMICS software. DOI: http://dx.doi.org/10.7554/eLife.14740.008

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Software

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: Ni-NTA Agarose (15 uL buffer equilibrated bead slurry) was incubated with 3 μM RAD52, 3 μM RPA in the presence or absence of 3 μM ‘1’ or ‘6’ , in the binding buffer (30 mM Tris-Acetate pH7.5, 1 mM βME, 150 mM KCl, 30 mM Imidazole, 5% glycerol, and 0.2% Nonidet P40 substitute). After 30 min incubation on a neutator at 4°C samples were spun down, and the aliquots of unbound ('free') proteins from each reaction were saved. Then the beads were washed and the bound proteins were eluted with 20 uL elution buffer (the same as the binding buffer, but with 400 mM Imidazole) and saved for gel electrophoresis. Free proteins and proteins co-eluted from the beads ('bound') were separated on the 12% SDS PAGE gel. Lane 1 is a loading control, which shows RAD52 and the three subunits of RPA (RPA70, RPA35, and RPA14). The proteins and the compounds present in each reaction are indicated in the table above the gel. The carton on the left of the gel schematically depicts the experiment: RAD52 protein binds to the Ni-NTA beads through the interaction with its 6xHis tag; RPA is untagged and can be retained on the beads only through a specific interaction with RAD52 . The experiment was repeated three times (a representative gel is shown) and no change in the ratio of RAD52 and RPA co-eluted from the beads in the presence and absence of ‘1’ or ‘6’ was detected. DOI: http://dx.doi.org/10.7554/eLife.14740.009

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Incubation, Binding Assay, Nucleic Acid Electrophoresis, SDS Page

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Three individual monomers of the RAD52-NTD undecameric ring (PDB 1KNO) are colored yellow, green and blue respectively. ‘1’ and ‘6’ occupy similar sites at the interface of two subunits. Two grey lines in each panel indicate the approximate boundaries of the ssDNA-binding groove. ( b ). MOE ligand maps highlight water mediated interactions as well as interactions with amino acids. ‘1’ likely mediates interactions through R55, V128, E140, and E145, as well as through water contacts made with G59, M56, and K141. ‘6’ likely binds via hydrogen bonding via D149 and I166 as well as through water interactions with E140, K144, and R153. DOI: http://dx.doi.org/10.7554/eLife.14740.011

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Binding Assay

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Representative images showing fields of cells from the comet assay for untreated, as well as from UCN01 (300 nM) and HU (2 mM) treated cells in the presence and absence of ‘1’, ‘6’ , and siRAD52. ( b) ‘1’ and ‘6’ at 1 or 25 μM recapitulate RAD52 depletion. GM1604 cells, transfected or not with siRNAs against RAD52, were treated as indicated, in the presence or absence of the inhibitor. At the end of treatment, DSBs were analyzed by neutral comet assay. Data are presented as the mean ± SEM from two independent experiments; p values are shown in the graph when differences are statistically significant. ( c – d ) Inhibitors ‘1’ and ‘6’ , respectively decrease the mean tail moment following HU treatment with IC 50 values ranging from mid nanomolar to low micromolar. Cells were treated as in ( b ). Data are from three independent experiments. DOI: http://dx.doi.org/10.7554/eLife.14740.012

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Single Cell Gel Electrophoresis, Transfection, Neutral Comet Assay

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Western blot analysis of BRCA2, RAD52, and GAPDH (loading control) protein levels in GM01604 cells treated with Ctrl, BRCA2, and RAD52 siRNAs. ( b ) Evaluation of cell death after replication stress in cells treated with ‘1’ . GM01604 cells were transfected with the RAD52 or BRCA2 siRNAs, alone or in combination, and 48h thereafter treated as indicated. The ‘1’ inhibitor or solvent (DMSO) was added to media 1h prior to replication stress. Cell viability was evaluated by LIVE/DEAD assay as described in the 'Materials and methods'. Data are presented as percentage of dead cells and are mean values from three independent experiments. Error bars represent standard error. The numbers shown in the graph represent the p value; the first p value of each pair refers to untreated cells while the second to the treated cells (2 way ANOVA). ( c ) Representative images: live cells are stained green, while dead cells are red. DOI: http://dx.doi.org/10.7554/eLife.14740.015

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Western Blot, Transfection, Live Dead Assay, Staining

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Docking workflow involved RAD52-NTD undecameric ring (PDB 1KNO) pre-processing, AnalytiCon Discovery MEGx Natural Products Screening Library pre-processing, and classical docking using the Dock utility of MOE. Top ranking poses (those with the lowest energy scores from the London dG scoring function) were subjected to a refining docking step involving force field-based energy minimization. From these complexes, predicted binding free energies were calculated. Workflow validation involved RAD52-NTD pre-processing, pre-processing of ‘1’ and associated decoys (DUD-E), followed by classical docking and score ranking. ROC curves were then generated and analyzed. Scores of the conformations of inhibitor compounds and of conformations of their respective decoy compounds were compared. ( b ) Docking scores (kcal/mol) for the individual conformations of compound ‘1’ and decoy compounds were binned and plotted as histograms. The low docking scores, as indicated by the more negative predicted free energies of ‘1’ when compared to decoys, indicate more favorable poses, and highlight a distinct separation between true positives and true negatives. ( c ) Receiving-operating characteristic (ROC) curve shows that the classifier used, i.e. the scoring function, was close to optimal in distinguishing compound ‘1’ conformations from those of decoys confirmed by AUC analysis yielding a value of 0.9973. ( d ) Electrostatic surface potential of three monomers of the RAD52-NTD undecameric ring (PDB 1KNO) depicting NP-004255 within the ssDNA binding groove. ( e ). MOE ligand maps highlight water mediated interactions with E145 and D149 as well as via hydrogen bonding with amino acids R55. DOI: http://dx.doi.org/10.7554/eLife.14740.016

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Refining, Binding Assay, Generated

Journal: eLife

Article Title: Small-molecule inhibitors identify the RAD52-ssDNA interaction as critical for recovery from replication stress and for survival of BRCA2 deficient cells

doi: 10.7554/eLife.14740

Figure Lengend Snippet: ( a ) Aromatic region of the 1D 1 H NMR spectrum of compound NP-004255 alone (black) and the WaterLOGSY spectrum of 40 μM compound NP-004255 in the presence of 3.3 μM RAD52 (red). The nonexchangeable proton peaks are labeled using atom names as indicated on the structure of compound NP-004255 . ( b ) IC 50 values for inhibition of ssDNA binding and wrapping were determined using FRET-based assays that follow the change in geometry of a Cy3-dT30-Cy5 substrate (black circles). The computed IC 50 value is shown above the curve. Titration of the RAD52-dsDNA with NP-004255 (grey boxes) shows that this inhibitor does not perturb the RAD52-dsDNA interaction. ( c ) Aromatic region of the 1D 1 H NMR spectrum of compound NP-004255 alone (black) and the WaterLOGSY spectrum of 40 μM compound NP-004255 in the presence of 3.3 μM RPA (red). ( d ) Titration of the RAD52-RPA- Cy3-dT 30 -Cy5 complex with NP-004255 (black circles). The computed IC 50 value is shown below the curve. Green squares show titration of the RPA- Cy3-dT 30 -Cy5 complex with NP-004255 indicating NP-004255 does not perturb the RPA-ssDNA interaction. DOI: http://dx.doi.org/10.7554/eLife.14740.017

Article Snippet: The 6xHis-tagged human RAD52 protein was expressed and purified as previously described , except a size exclusion chromatography (HiPrep 16/60 Sephacryl S-300 HR GE Healthcare Life Sciences, Pittsburgh, PA, USA) step was added between the heparin and Resource S columns to remove low molecular weight impurities.

Techniques: Labeling, Inhibition, Binding Assay, Titration

Journal: International Journal of Molecular Sciences

Article Title: Current State of Knowledge Regarding WHO High Priority Pathogens—Resistance Mechanisms and Proposed Solutions through Candidates Such as Essential Oils: A Systematic Review

doi: 10.3390/ijms24119727

Figure Lengend Snippet: Studies assessing the antimicrobial activity of essential oils against methicillin resistant, vancomycin-intermediate and -resistant S. aureus (a non-exhaustive list).

Article Snippet: Khamis AS et al., 2021 , MRSA ATCC 33591 E. coli NCTC 10418 P. aeruginosa NCTC 10662 Bacillus subtilis ATCC 6059 Micrococcus luteus ATCC 9341 S. aureus NCTC 6571 , Juniperus chinensis L. (Chinese juniper) Juniperus seravschanica Kom. (Pashtun juniper) versus their methanolic crude extracts , Disc diffusion , Although all EOs tested showed antimicrobial activity, increased activity was observed against M. luteus and B. subtilis . Only the crude methanolic extracts manifested an increased antibacterial activity against MRSA and S. aureus . A higher antimicrobial activity was observed for methanolic crude extract of J. seravschanica (from Oman) against MRSA. , [ ] .

Techniques: Activity Assay, Diffusion-based Assay, Inhibition, Cytotoxicity Assay, Electron Microscopy, In Vitro, Dilution Assay, Preserving, Quantitative Proteomics, Nucleic Acid Electrophoresis, Membrane, Confocal Laser Scanning Microscopy, Concentration Assay, Titration, Bacteria, Microscopy, Transmission Assay, Microdilution Assay, Produced, Modification, Clinical Proteomics, Blocking Assay, Staining, Cell Culture, Fourier Transform Infrared Spectroscopy, Spectroscopy, Reverse Transcription, Real-time Polymerase Chain Reaction, Crystal Violet Assay, Expressing, Flow Cytometry, In Vivo, Liposomes, Time-Kill Assay, Formulation, MTT Assay, Incubation, Thin Layer Chromatography, Bioassay, Antibiofilm Assay, Resazurin Assay, Biofilm Production Assay, Control, Infection, Cream, Antioxidant Activity Assay, Permeability, Virus, Extraction, Isolation

Journal: Journal of Neuroinflammation

Article Title: Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica

doi: 10.1186/s12974-016-0767-4

Figure Lengend Snippet: Human complement (HC) is toxic to mouse neurons and oligodendrocytes in monocultures. IncuCyte live imaging of neurons, mature oligodendrocyte (OL), and oligodendrocyte progenitors (OPC) monocultures treated with HC (2% in neurons and 5% in OLs and OPCs) for 4 h. DRAQ7 was added in the culture medium to label dead cells. a , c , and e Phase contrast images show DRAQ7 staining ( red ) in the absence (CTRL) and presence (HC) of human complement. b , d , and f The percentage of DRAQ7+ neurons ( b ), OLs ( d ), and OPCs ( f ) increased rapidly following exposure to HC. g and h Astrocytes (AST) exposed to 5% HC for 4 h showed no evidence of cytotoxicity as measured and quantified by IncuCyte. CTRL: PBS. Scale bars 100 μm

Article Snippet: Each well was scanned with a 10× objective lens in nine randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the following filter sets: 460/524 nm (green fluorescence, to detect enhanced green fluorescent protein, eGFP) and 585/635 nm (red fluorescence, to detect the DRAQ7 dead cell nuclei; Cell Signaling Technology, Danvers, MA).

Techniques: Imaging, Staining

Journal: Journal of Neuroinflammation

Article Title: Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica

doi: 10.1186/s12974-016-0767-4

Figure Lengend Snippet: Mixed cultures of neurons, oligodendrocytes, and astrocytes demonstrate reduced complement cytotoxicity. Neuroglial mixed cultures prepared from PLP-eGFP mouse pups were treated with 5% HC for 4 h in the presence of DRAQ7 and imaged using IncuCyte. a Phase contrast images of IncuCyte cultures treated with human complement (HC) at 0 and 4 h. Insert : Fluorescence image showing oligodendrocytes (PLP-eGFP; green ) and dead cells (DRAQ7; red ). Arrows : neurons. Arrow heads : astrocytes. b Quantitation of the percentage of DRAQ7+ neurons at 2 and 4 h in the presence of 2% or 5% HC. c Fluorescence image and quantitation of the percentage of PI+ dead neurons following exposure to 5% HC for 4 h. Left : fluorescence image of mixed cultures exposed to CTRL or HC and stained with PI ( red ) followed by NeuN ( green ) at 4 h. Note the dead neurons (PI+/NeuN+) in the HC-treated cultures. d , e IncuCyte fluorescence images ( left ) and quantitation of ( d ) differentiated oligodendrocyte (OL, arrows ) and ( e ) oligodendrocyte progenitor (OPC) death in mixed cultures. OPCs and differentiated OLs were defined by morphology. More than 150 differentiated oligodendrocytes (OL) were counted for each experiment. CTRL: PBS. Scale bars 100 μm

Article Snippet: Each well was scanned with a 10× objective lens in nine randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the following filter sets: 460/524 nm (green fluorescence, to detect enhanced green fluorescent protein, eGFP) and 585/635 nm (red fluorescence, to detect the DRAQ7 dead cell nuclei; Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Quantitation Assay, Staining

Journal: Journal of Neuroinflammation

Article Title: Variable sensitivity to complement-dependent cytotoxicity in murine models of neuromyelitis optica

doi: 10.1186/s12974-016-0767-4

Figure Lengend Snippet: Kinetics of neuron, OL, and OPC loss in the presence of NMO rAb #53-mediated astrocyte injury. a Fluorescence (left) and phase contrast IncuCyte (right) images of neuroglial mixed culture treated with rAb #53 plus 5% HC in the presence of DRAQ7 (PLP-eGFP oligodendrocytes [green] and DRAQ7 dead cells [red]). Arrows : neurons. Arrow heads : astrocytes. Circled area : focal region of astrocyte depletion. b , c , d , and e Quantitation of the percentage of DRAQ7+ AST ( b ), neuron ( c ), OL ( d ), and OPC ( e ) in the mixed cultures. Cells were identified as described in the . Statistical analyses were performed by multiple unpaired Student’s t test. ** p < 0.01, n > 3. Scale bars 50 μm

Article Snippet: Each well was scanned with a 10× objective lens in nine randomly selected positions at 15 min intervals with high definition phase contrast and epifluorescence microscopy using the following filter sets: 460/524 nm (green fluorescence, to detect enhanced green fluorescent protein, eGFP) and 585/635 nm (red fluorescence, to detect the DRAQ7 dead cell nuclei; Cell Signaling Technology, Danvers, MA).

Techniques: Fluorescence, Quantitation Assay

Journal: Transplant International

Article Title: Assessment of Mitochondrial Respiration During Hypothermic Storage of Liver Biopsies Following Normothermic Machine Perfusion

doi: 10.3389/ti.2024.12787

Figure Lengend Snippet:

Article Snippet: Briefly, biopsies were incubated with the fluorescent stains Hoechst 33,342 (blue, staining all nuclei, Molecular Probes; final concentration 5 μg·mL −1 ), propidium iodide (PI; red, staining the nuclei of dead cells, Molecular Probes; final concentration 500 nM), and wheat germ agglutinin (WGA; green, staining of cell morphology, Molecular Probes, Eugene, OR, United States; 5 μg·mL −1 final concentration) at room temperature for 15 min.

Techniques: Control, Transplantation Assay, Phospho-proteomics, Confocal Microscopy, Titration