Journal: The Journal of Cell Biology

Article Title: Rad51-mediated replication fork reversal is a global response to genotoxic treatments in human cells

doi: 10.1083/jcb.201406099

Figure Lengend Snippet: RAD51 is present at forks upon mild genotoxic stress and modulates fork progression and integrity. (A) Linear regression analysis shows strict direct correlation (P < 0.0001) between accumulation of ssDNA at the fork (median values of ssDNA regions at the junction) and frequency of fork reversal. Results from two independent experiments are displayed for untreated U2OS cells and for each genotoxic treatment. (B) HEK293T cells were EdU-labeled as indicated in Fig. S5 C and treated with sublethal doses of genotoxic drugs (0.5 mM HU, 200 nM MMC, or 25 nM CPT). Proteins and relative posttranslational modifications associated with replication forks were isolated by iPOND procedure and detected with the indicated antibodies. The thymidine (Thy; 10 µM) chase experiment is used to discriminate proteins associated with chromatin behind replicating forks. In the control (Ctrl) experiment, the click reaction is performed using DMSO instead of biotin azide. 1 µM CPT treatment is used as positive control to induce high replication stress and DSBs. (C) Immunofluorescence staining for U2OS cells grown on coverslips and treated with the indicated drugs for 1 h. Red staining, RAD51; green staining, EdU; blue, DAPI. Bar, 15 µM. (D) DNA fiber spreading. Statistical analysis of IdU replicated track length in U2OS cells, comparing not treated (NT) conditions with the indicated treatments. U2OS cells were transfected with siRNA against luciferase (siLuc) or RAD51 (siRAD51) 24 h before CldU or IdU labeling. At least 100 tracks were scored per sample. Horizontal lines represent the median value, and boxes and whiskers indicate 10–90th percentiles. Statistical analysis: one-way ANOVA; ns, not significant; ***, P ≤ 0.001. (E) PFGE analysis for DNA breakage detection in untreated U2OS cells and upon 1-h treatment with indicated doses of genotoxic treatments. U2OS cells were transfected with siRNA against luciferase or RAD51 24 h before treatments. 1 µM camptothecin (CPT) treatment is used as a positive control for DSB formation. The graph shows quantitative DSB induction from three independent experiments and includes average value and standard deviations (error bars). Statistical analysis: two-way ANOVA; ns, not significant; *, P ≤ 0.05.

Article Snippet: Electrophoresis was performed for 21 h at 14°C in 0.9% (wt/vol) Pulse Field Certified Agarose (Bio-Rad Laboratories) containing Tris-borate/EDTA buffer in a PFGE apparatus (CHEF DR III; Bio-Rad Laboratories), according to the following protocol (block I: 9 h, 120° included angle, 5.5 V/cm, 30 to 18-s switch; block II: 6 h, 117° included angle, 4.5 V/cm, 18 to 9-s switch; block III: 6 h, 112° included angle, 4.0 V/cm, 9 to 5-s switch).

Techniques: Labeling, Isolation, Control, Positive Control, Immunofluorescence, Staining, Transfection, Luciferase

Journal: Applied and Environmental Microbiology

Article Title: Characterization of Vibrio fluvialis -LikeStrains Implicated in Limp LobsterDisease

doi: 10.1128/aem.69.12.7435-7446.2003

Figure Lengend Snippet: FIG. 3. Electrophoretic migration patterns and similarity dendrogram of NotI-digested Vibrio fluvialis-like genomic DNA obtained by PFGE. Lanes 1 to 18 represent strains 1AMA, 6A1MA, 15A4TSA, DB6, DB8, 2E1MA, 3DMA, 4D119MA, 5E2MA, 8D122MA, 9DMA, 10C119MA, 11EMA, 18C110MA, DB7, 27F3MA, 28F4MA, and 31F7G, respectively. Each strain’s PFGE pattern type is listed as well. DNA molecular weight scale was derived from XbaI-digested Salmonella newport AM01144 genomic DNA. V. fluvialis-like strain 7E1AMA is not shown in the picture but was shown to be a member of the PFGE subtype type B group by PFGE analysis.

Article Snippet: The DNA in the gel was resolved using a Bio-Rad CHEF-DRII PFGE apparatus (Bio-Rad Laboratories, Hercules, Calif.).

Techniques: Migration, Molecular Weight, Derivative Assay

Journal: Journal of applied microbiology

Article Title: Characterization of closely related lactococcal starter strains which show differing patterns of bacteriophage sensitivity.

doi: 10.1046/j.1365-2672.2003.02134.x

Figure Lengend Snippet: Fig. 1 Pulsed-field gel electrophoresis patterns of Sma I digests of genomic DNA from Lactococcus lactis subsp. lactis strains. Lanes 1–10, strains LW2004, LW2006, LW1999, LW2003, LW2005, LW2000, LW2001, LW2002, LW2515, IL1403. The pulse time used was 1–30 s

Article Snippet: DNA embedded in agarose was digested with Sma I (New England Biolabs, Beverly, MA, USA), loaded into the wells of 1% agarose gels (pulsed-field certified agarose; BioRad Laboratories, Hercules, CA, USA), and separated at 200 V for 20 h at 14 C in 0Æ5X Tris-borate buffer using a CHEF DR II PFGE apparatus and model 1000 mini chiller (Bio-Rad).

Techniques: Nucleic Acid Electrophoresis

Journal: Journal of Clinical Microbiology

Article Title: Multilocus Sequence Typing for Characterization of Methicillin-Resistant and Methicillin-Susceptible Clones of Staphylococcus aureus

doi: 10.1128/jcm.38.3.1008-1015.2000

Figure Lengend Snippet: FIG. 2. PFGE of pairs of isolates with identical allelic profiles. Chromosomal DNA from pairs of isolates of STs 25 (lanes b and c), 30 (lanes d and e), 34 (lanes f and g), 36 (lanes h and i), 39 (lanes j and k), 45 (lanes l and m), 47 (lanes n and o), and 49 (lanes p and q) were digested with SmaI and were separated by PFGE. Concatenated bacteriophage lambda molecular size markers were run in lanes a and r. Numbers on the right are in kilobase pairs.

Article Snippet: The samples were run on 1% agarose gel in 0.5% TBE buffer (44.5 mM Tris, 44.5 mM borate, 1 mM EDTA) on a CHEF DR-III PFGE system (Bio-Rad, Hemel Hempstead, Hertsfordshire, United Kingdom) by using an initial switching time of 1 s which was increased to 5 s for 12 h, followed by 12 h with an initial switching time of 15 s which was increased to 30 s, by using a voltage of 6 V cm21 (3).

Techniques: