Gene expression.

<p>Gene expression in preadipocytes (denoted Undiff) and differentiated human preadipocytes in 2D aligned and random fibers Analyzed data is ΔCt values from technical duplicates. A) PCA analysis of the variation between samples. B) A heatmap was created from mean expression levels in three donors, and the expression profiles were analyzed with hierarchical clustering (UPGMA method with Euclidian method for distance measure).</p

Lipolysis in differentiated and mature adipocytes.

<p>Lipolysis was measured as glycerol release and normalized between 0 and 100% to determine EC50 values for human preadipocytes differentiated in 2D (black, solid line), n = 18 differentiated at three separate occasions, human preadipocytes differentiated in aligned (green line), n = 18 differentiated at three separate occasions and human preadipocytes differentiated in random fibers (magenta line), n = 18 differentiated at three separate occasions, mature adipocytes (black dashed line) measured as mean of triplicates isolated from 5 different subjects. A) All four data sets normalized to min 0 and max 100% and fitted to the same slope, no significant differences between the EC50s. B) All four data sets normalized to min 0 and max 100% and fitted to individual, three parameter slopes. C) Fully differentiated human preadipocytes (N = 3, n = 6) or human mature adipocytes (N = 7) were lysed levels of HSL was measured with western blot as arbitrary units normalized to loading control actin. P-value <0.05 is indicated with *, bars or lines without a star were not found to be significantly different from any other bar in the grap.</p

Human adipose derived stem cells growing in PCL fiber matrices.

<p>A) Normalized intensities of the CARS (2845 cm⁻¹) signal of the fibers (violet, solid) and the MPEF signal (495–530 nm) (black, dashed lines, one line for each cell) of the cells in the matrices in the z dimension for the aligned (left) and random (right) matrices. 5 cells were investigated for each condition, for the random matrix two fibers were shown to visualize the different fiber profiles in this matrix. B) 3D reconstructions of the MPEF signals of the cells on/in the fiber matrices for aligned (left) and random (right) matrices. Nuclei are indicated with circles and fibers with arrowheads (scalebar: 30 µm).</p

Proliferation of human adipose derived stem cells.

<p>P-values are indicated with * when p<0.05 and *** when p<0.001. 2D (black), aligned (green) and random (magenta). A) Proliferation was measured by nuclear count at the indicated time points during the differentiation, in wells seeded with 2D (56250 c/cm²), aligned (112500 c/cm²) and random (112500 c/cm²), n = 10. B) Number of cells was measured as in 1A. C) Live-dead images.14-days differentiated cells were stained live cells (calcein AM) indicated with green and dead cells (Ethidium homodimer) indicated with red thenimaged.</p

Lipid accumulation during adipogenic differentiation of human preadipocytes in 2D and fiber matrixes.

<p>P-values are indicated with ** when p<0.01 and *** when p<0.001, bars without a star were not found to be significantly different from any other bar in the grap. Mature adipocytes (open bars), 2D (black), aligned (green) and random (magenta). A) Upper: 14 days differentiated cells were stained with oil-red-o and imaged in a bright field microscope. Lower: Lipid accumulation was measured as absorbance of eluted oil-o-red at 490 nm at the differentiation day 2 and 14 and normalized to the cell number counted using Cellavista after staining with Hoechst nuclear dye, n = 10. B) Upper: 14 days differentiated cells unstained imaged with CARS (red signals) and MPEF (green signals) microscopy (Order of the images: 2D, aligned, random; scalebar: 30 µm). Lower: Size distribution of lipid droplets in the adipocytes. Presented is the distribution between 6 and 14 µm in 1 µm bins. 30 cells were evaluated per condition. The total droplet number evaluated in each condition was set to 100%. C) Approximation of the droplet size distribution with a Lorentizian function from the data set in 2B. Each data set was normalized to the total number of counted lipid droplets per well in each condition. D) Levels of perilipin in fully differentiated human preadipocytes (N = 3, n = 6) and primary, mature adipocytes (N = 7) measured as a.u. with western blot and normalized to actin content and to a standard as described in the <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113620#s2" target="_blank">methods</a> section.</p

Fitting parameters for the Lorentizian distribution in Figure 3C.

<p>Fitting parameters for the Lorentizian distribution in <a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0113620#pone-0113620-g003" target="_blank">Figure 3C</a>.</p

Glucose uptake in differentiated and mature adipocytes.

<p>Insulin stimulated glucose transport was measured as uptake of deoxy-D-glucose 2-[1- ¹⁴C] in response to indicated doses of insulin during 60 min and normalized between 0 and 100% glucose uptake and outliers were excluded using the tukey test in human preadipocytes differentiated in 2D (solid black line, black dots), n = 19 differentiated at three different occations human preadipocytes differentiated on aligned matix (green line and dots), n = 18 differentiated at three different occations, human preadipocytes differentiated on random matrix (magenta line and dots), n = 17 differentiated at three different occations, human mature adipocytes (dashed black line, open circles) measured as a mean of duplicates isolated from 7 different subjects. A) All four data sets normalized to min 0 and max 100% and fitted to the same slope, inserted: EC50 values from the dose-responses, statistical testing was performed using students t-test, * denotes a p-value lower than 0.05. B) Glucose uptake, basally and stimulated with 100 nM insulin, normalized to total protein content. C) All four data sets normalized to min 0 and max 100% and fitted to individual, three parameter slopes. D) Insulin receptor levels measured as arbitrary units and normalized to actin content with western blot. Differentiated human preadipocytes(n = 6, N = 3) or human mature adipocytes N = 7 E) Fully differentiated human preadipocytes N = 3 or human mature adipocytes, N = 6, were stimulated with indicated concentrations of insulin for 10 minutes subjected to SDS-PAGE and western blot, phosphorlylation of serine 473 of PKB measured as arbitrary units and normalized to actin content and maximal phosphorylation was set to 100%.</p