Journal:

Article Title: Pituitary adenylate cyclase-activating polypeptide is an autocrine inhibitor of mitosis in cultured cortical precursor cells

doi:

Figure Lengend Snippet: Characterization of E13.5 cortical precursor cultures. Cells were incubated for 3 (a, b, d, e) or 24 h (c and f) and examined by phase (a and d) or brightfield microscopy to detect neuronal markers, immunoreactive MAP2 (b and c), and NSE (e and f). Neuronal markers increase from ≈50% at 3 h (b and e) to ≈90% at 24 h (c and f) (positive staining indicated by arrows; arrowhead = negative cells). Cells were unstained when primary antibody was omitted. MAP2 and NSE analyses were performed on five and two experiments, respectively. (Bar = 50 μm.)

Article Snippet: After 4% paraformaldehyde fixation (20 min), immunocytochemistry was performed with antibodies to MAP2 (1:1,000; I. Fischer, Medical College of Philadelphia), NSE (1:1,000; Polysciences), glial fibrillary acidic protein (1:1,000; Dako), RC1 (R. Schwarting, Dana–Farber Cancer Institute), myelin basic protein (1:500; D. Colman, Mt.

Techniques: Incubation, Microscopy, Staining

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 1. Design of the 4HB DNA NS structure. (a) A cylinder model of the 4HB square lattice (see cross-section). Each cylindrical rod represents a single DNA double helix. Two insertion and deletion sites that produce a superhelical strain are shown in blue and red, respectively. (b) CanDo, a 3D DNA origami structure prediction server,24 predicted a coil structure. Single-stranded DNA (ssDNA) handles projecting out from both ends are shown as red and orange lines, respectively. The complementary ssDNA antihandles are modified with adhesive peptide (cRGDfK, blue hexagon) to the extracellular domain of integrins and biotin (black circle) to attach the NS to the bottom of the dish. (c) An AFM image of the 4HB-NS. The shape was flexible enough to measure single integrin traction force but also showed repetitive bending or expected loops. Scale bar, 200 nm.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Modification, Adhesive

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 2. Visualizing NS extensions by single integrins. (a) A typical fluorescence image of Cy3 NS (red) and GFP-paxillin (white) expressed in HFFs and observed by TIRFM. The boxed area is expanded in (b). Scale bar, 10 μm for (a) and 2 μm for (b). The right cartoon in (b) shows NSs attached with integrins via cRGDfK and the glass surface via the biotin−avidin system. (c) A histogram of flattening NS fluorescence spots located outside the cells. The flattening was calculated as 1 −σshort/σlong, where σshort and σlong, respectively, denote the standard deviation for the short and long axes when the fluorescence spot was fit to a 2D Gaussian function. (d, e) Histograms of flattening NS fluorescence spots within (d) and outside FAs underneath cells (e). (n = 370, 428, and 292 molecules from 6, 6, and 5 cells for (c, d, e), respectively.) (f, g) Time trajectories of the angle between the long axis of NS and the horizontal direction of the picture for the spots undergoing Brownian motion (f) and stretching within FAs (g). Each color represents an individual NS.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Fluorescence, Avidin-Biotin Assay, Standard Deviation

Journal: ACS nano

Article Title: A Programmable DNA Origami Nanospring That Reports Dynamics of Single Integrin Motion, Force Magnitude and Force Orientation in Living Cells.

doi: 10.1021/acsnano.2c12545

Figure Lengend Snippet: Figure 5. NS force sensor indicated dynamics of loaded force and single integrin motions in living cells. (a−c) Representative traces showing the length of the NS (top), loaded force on the NS (middle), and angle of the NS (bottom) for an integrin-bound NS. The force trace was calculated from the NS length. The thin lines indicate raw data; the bold lines indicate moving averages. (d, e) The colors of the traces correspond to (a−c), and the gradations of the colors correspond to the time course. Representative traces showing a single integrin motion tethered with an NS (d) and the force vector (e) in polar coordinates. Colors correspond to (a−c). (f) Histograms of the loaded force distributions for cRGDfK-labeled NS (red, n = 59 molecules; N = 23 cells) and cRGDfK-less NS (blue, n = 138 molecules; N = 24 cells) underneath the cells.

Article Snippet: For positive control experiments (cell adhesion with biotinylated cRGDfK), after neutravidin was washed out, 200 μL of biotinylated cRGDfK (1−150 nM in DNA origami buffer, Peptides International, PCI-3697-PI) was added on the glass-bottomed dish, and the mixture was incubated for 10 min. Unbound biotinylated cRGDfK was washed out using a DNA origami buffer.

Techniques: Plasmid Preparation, Labeling