Table 1.
Primary antibodies used for Western blot analysis.
Fig 1.
Sudan III staining for lipid detection in HMGEC.
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.
Fig 2.
Ultra-structural analysis of HMGEC.
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.
Fig 3.
A. Electric Cell-substrate Impedance Sensing (ECIS) of HMGEC over 120 hours. Cells were grown in serum-free medium for 48 hours before incubation for another 72 hours in serum-free medium (SFM, black) or serum-containing medium (SCM, red). B. There was significantly increased impedance after 1d culture with SCM compared to SFM (* p ≤ 0.05, student´s t-test, n = 4). C. Real-time RT-PCR analyses of desmoplakin 1/2 mRNA expression in HMGEC. Cells were grown to 90% confluence followed by either cultivation in serum-free or serum-containing medium for 1 day. The fold increase transcript levels are shown as mean ± SEM and statistical significance vs. control is indicated by asterisks (n = 4, student´s t-test; * p < 0.05). D. After switching to SCM cellular morphology was changed. Previously distributed cells formed groups and cell contacts. Magnification: 10x.
Fig 4.
Expression of cytokeratins (CK) and GAPDH in HMGEC.
(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; * p < 0.05).
Fig 5.
Quantitative analysis of lipid profile of HMGEC cultivated for 1 day or 3 days in serum-containing medium.
Values are shown as the mean of 12 measurements ± SEM (n = 15). Phosphatidylcholine (PC), cholesterol (Chol), phosphatidylserine (PS), phosphatidylethanolamine (PE), cholesterol ester (CE), sphingomyelin (SM), diacylglycerol (DAG), triacylglycerol (TAG), ceramide (Cer) and wax ester (WE) were detectable. (Student´s t-test; * p ≤ 0.001)
Fig 6.
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).
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.
Fig 7.
Ultra-structural analysis of HMGEC after 1 day stimulation with serum-containing medium supplemented with 100 μM EPA (A) and 20% FCS (B).
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).
Table 2.
Comparison of lipids reported in the current study to those reported for HMGECs [4] and epithelial cells [37] in previous reports.