3D reconstruction by digitized and computer based processed histological sections of the pig lacrimal gland.

<p>A) Visualization of seven excretory lacrimal ducts (different colors) within the paraffin embedded lacrimal gland (grey). B) Separate exposure of each lacrimal duct in different colors with surrounding gland tissue reconstruction. Only the yellow-marked duct system is displayed alone, without gland tissue reconstruction.</p

Excretory ducts of the left lacrimal system in pig.

<p>Conjunctival view at the level of the eye angle, left scale bar: 2 mm. EB: eyeball; LEA: left eye angle; 1–6: excretory ducts. Right bottom square: higher magnification of an excretory duct, scale bar: 200 µm.</p

Expression of cytokeratins (CK) and GAPDH in HMGEC.

<p>(A) Representative western blots show specific bands for GAPDH, CK1, CK5, CK6 and CK14. (B) Intensities (normalized to GAPDH) are shown as mean ± SEM and statistical significance vs. 21d serum treatment is indicated by asterisks (n = 6, one-way ANOVA; * <i>p <</i> 0.05).</p

Comparison of lipids reported in the current study to those reported for HMGECs [4] and epithelial cells [37] in previous reports.

<p>phosphatidylethanolamine (PE), phosphatidylserine (PS), phosphatidylcholine (PC), sphingomyelin (SM), diacylglycerol (DAG), ceramide (Cer), cholesterol ester (CE), free cholesterol (Chol), and wax ester (WE)</p><p>Comparison of lipids reported in the current study to those reported for HMGECs [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128096#pone.0128096.ref004" target="_blank">4</a>] and epithelial cells [<a href="http://www.plosone.org/article/info:doi/10.1371/journal.pone.0128096#pone.0128096.ref037" target="_blank">37</a>] in previous reports.</p

Sudan III staining for lipid detection in HMGEC.

<p>Cells were either cultured in serum-free medium until they reached 70–90% confluence (A, B) or cultured in serum-containing medium for 1 (C), 7 (D) or 14 (E) days. The graph in (F) shows quantification of Sudan III stained areas normalized to the cell count per image. Serum-free treated cells showed no lipid accumulation. Cells in serum-containing media accumulated lipids in the cytoplasm after one day treatment. Lipid accumulation decreased over time. The red stain in the pictures indicates lipid droplets.</p

Ultra-structural analysis of HMGEC.

<p>Cells were either cultured in serum-free medium until they reached 90% confluence (A) or cultured in serum-containing medium for 1 (B, higher magnification in F), 3 (C), 7 (D) or 14 (E) days. Cytokeratin filaments (CF) elongated and desmosomes (black arrow heads) increased in serum-treated cells over time, but desmosomes were not visible in cells grown in serum-free media. Lipid droplets (arrows) can be seen in serum-treated cells. N = nuclei, ES = extracellular space.</p

A. Sudan III staining to visualize lipid accumulation of HMGEC after 1 day or 7 days stimulation with addition of 10% FCS (C, D), high glucose (E, F), lipid cocktail (G, H), 100μM EPA (I, J) in10% serum-containing medium (A, B) or sebomed medium (K, L).

<p>Fig 6M shows quantification of Sudan III stained areas normalized to the cell count per image. In general, lipid accumulations were more prominent after 1 day compared to 7 days cultivation in serum-containing medium. Highest levels of lipids were visible after 1 day treatment with 100μM EPA (I). Red stain indicates lipid droplets.</p

Ultra-structural analysis of HMGEC after 1 day stimulation with serum-containing medium supplemented with 100 μM EPA (A) and 20% FCS (B).

<p>Cytokeratin filaments (CF), desmosomes (black arrow heads) and lipid droplets (arrows) are visible in 20% FCS treated cells. EPA stimulated cells show numerous lysosomes (white arrow heads).</p