Journal: Nucleic Acids Research

Article Title: Visualizing helicases unwinding DNA at the single molecule level

doi: 10.1093/nar/gkq173

Figure Lengend Snippet: DNA unwinding by AddA N B N is characterized by pauses and bursts of activity. ( A ) Example of how a single time-course can be segmented into pauses and unwinding phases. The raw intensity data (light blue open circle) were smoothed by running average using an optimum window size (shown as pink and dark blue lines). Then, the first derivative at every time point was calculated (grey open circle). If the derivative trace (grey open circle) is smoothed using an median filter (black line), then the histogram of the smoothed derivative values (inset) reveals two populations, one corresponding the pauses (clustered around zero) and one to the unwinding phases (clustered around 17 cpp). Thresholding ( Thr. ) of the derivative data (dashed line) allows the original intensity trace to be segmented into pauses (< Thr. , pink lines) and unwinding phases (> Thr. , dark lines). ( B–E ) To automatically analyze all data, a custom-written PERL algorithm was used (see ‘Materials and methods’ section). Using this algorithm, the distributions of the number of pauses per event (B), the duration of pauses (C), maximum rate of unwinding (D) and duration of unwinding phase (E) were obtained for 1 mM ATP (circle) and 3 μM ATP (triangle). All distributions are presented as percentage frequency, normalized to the maximum.

Article Snippet: To identify and characterize the pauses and unwinding phases a custom-written PERL algorithm (ActiveState Software Inc.) was developed.

Techniques: Activity Assay

Journal: PLoS ONE

Article Title: Functional Characterization of Germline Mutations in PDGFB and PDGFRB in Primary Familial Brain Calcification

doi: 10.1371/journal.pone.0143407

Figure Lengend Snippet: (AB) PDGF-BB—induced membrane ruffling of stably transfected PAE cells expressing mutant PDGF-Rβ receptor. (A) Fluorescent labeling of the actin cytoskeleton in PAE cells after 30 minutes of PDGF-BB exposure. Arrowheads indicate peripheral membrane ruffles. Cyan: DAPI, green: Alexa 488-conjugated phalloidin. Error bar: 30 μm. (B) The total amount of ruffles was counted in 20 fields per condition, and normalized over the total amount of cells. * p ˂0.05 as compared to wild-type PDGFRB -expressing PAE cells. Empty vector (pcDNA) and KD PDGFRB -transfected cells were used as negative controls. (CD) Wound healing assays of stably transfected PAE cells expressing different mutant PDGFRβ receptors. Confluent monolayers of PAE cells expressing different PDGFRB constructs were scratched using the WoundMaker™ and wound closure was monitored automatically every hour for 13 hours with the IncuCyte Zoom®. (C) Representative images of the wound at 0 and 13 hours of PDGF-BB stimulation. (D) Quantification of the increase in relative wound density within 13 hours. Error bars indicate standard deviation between 6 individual scratches, 2 images per scratch.

Article Snippet: This metric is calculated by custom algorithms, which are part of the IncuCyte software package (Essen BioScience).

Techniques: Membrane, Stable Transfection, Transfection, Expressing, Mutagenesis, Labeling, Plasmid Preparation, Construct, Standard Deviation