Journal: Nature Communications

Article Title: Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix

doi: 10.1038/ncomms9026

Figure Lengend Snippet: NIH 3T3 fibroblasts were seeded on Fn-coated glass with medium containing fluorescently labelled Fn (Alexa 488) in the absence ( a – f ) or presence ( g – l ) of 50 μg ml −1 ascorbic acid, and were cultured for 1 or 3 days, then fixed and immunostained for collagen type I. Collagen matrix production was greatly reduced in the absence of supplemental ascorbic acid. Images are z-projections of representative confocal z-stacks selected from five independent experiments. The third column of images shows the Fn (green) and collagen (red) images overlaid. Bar=20 μm.

Article Snippet: Cell culture and matrix manipulations NIH 3T3 fibroblasts (American Type Culture Collection, ATCC) were cultured in DMEM (ATCC) supplemented with 10% newborn calf serum (Gibco, Invitrogen) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: Cell Culture

Journal: Nature Communications

Article Title: Mechanical forces regulate the interactions of fibronectin and collagen I in extracellular matrix

doi: 10.1038/ncomms9026

Figure Lengend Snippet: NIH 3T3 fibroblasts were allowed to incorporate small amounts of Fn-FRET into their matrices for 3 days in the absence ( a ) or presence ( b ) of 50 μg ml −1 ascorbic acid. ( a – b ) Single z-slice images from the specified sample, colour-coded to reflect the calculated FRET ratios (see colour bar on right). ( c ) Representative normalized histograms of the FRET ratio distributions compiled from eight measurements for each treatment from a single experiment (black, no ascorbic acid; red, with ascorbic acid). The Fn-FRET denaturation points for the Fn monomer in 1 M GdnHCl and for the Fn dimer in 4 M GdnHCl points (see ) are indicated with coloured, vertical lines. Fn fibres with FRET ratios less than at the 1 M GnHCl calibration point (for Fn in solution) indicate partial secondary/tertiary structure loss due to unfolding. ( d ) Median FRET ratios from four independent experiments. Grey points represent individual measurements (23 for samples without and 25 for samples with ascorbic acid); black squares represent means (error bars, s.d.). ( e ) The same data as in panel ( d ) plotted as the mean (±s.d.) per cent unfolding of each group. Per cent partial Fn unfolding is the percentage of pixels with FRET ratios less than that seen at the 1 M denaturation curve point. Samples with and without ascorbic acid had significantly different median FRET ratios ( P <0.01) and mean per cent unfolding values ( P <0.05) (ANOVA, followed by a Tukey–Kramer post-hoc test were used because these data were analysed with additional groups shown in ). Bar=20 μm.

Article Snippet: Cell culture and matrix manipulations NIH 3T3 fibroblasts (American Type Culture Collection, ATCC) were cultured in DMEM (ATCC) supplemented with 10% newborn calf serum (Gibco, Invitrogen) in a humidified incubator at 37 °C with 5% CO 2 .

Techniques: