Figure 4
Effect of TNC on FN expression and assembly. (a) Representative western analysis of FN expression in total cell extracts of HUVECs cultured for 48 h on TNC-coated, BSA-pretreated or non-coated dishes. Equal amounts of protein (40 µg) were loaded in each lane and α-tubulin was used as loading control. Fold change over NC (normalized to α-tubulin) is shown below cropped blot. Full-length blots are shown in Fig. S7. (b) QPCR analysis of FN mRNA expression levels in different endothelial cells (left, HUVEC; HMEC, middle and HMVEC-d, right) cultured on indicated substrates in comparison to a non-coated (NC) surface (c). Time course (12–72 h) expression of FN (left), Axin2 (middle) and DKK1 mRNA (right) in HUVECs on TNC coating, non-coated or BSA-pretreated dish was monitored by QPCR. Fold change over NC (equal to 1, not shown on the graphs) was calculated using the ΔΔCt method (±S.D., n = 3). (d) Immunofluorescence staining of cortactin (white or green), filamentous actin (red) and nuclei (blue) of cells plated for 48 h on non-coated or TNC-coated coverslips. The zoomed area is indicated in white dotted square (right). Bar = 50 µm, and 10 µm in zoomed area. (e) Representative western blot analysis of cofilin phosphorylation at Ser 3 in total cell extracts of HUVECs cultured for 48 h on TNC-coated or non-coated dishes. Equal amounts of protein (40 µg) were loaded in each lane and ERK1/2 was used as loading control. Fold change over NC (normalized to ERK1/2) from 3 independent experiments is shown next to cropped blots. Full-length blots are shown in Fig. S7. (f) Immunofluorescence staining (wide-field images) of total FN in HUVECs (top) and HMECs (bottom) after 48 h of culture on non-coated or TNC-coated coverslips. FN fiber organization was analysed with homemade software coded in Matlab. Representative color-coded images of FN fibres in endothelial cells grown on indicated substrates are shown next to wide-field images. Color represents length of the fibres in µm (blue to red). At least 10 images were quantified for each condition and cumulative distribution of correlation length for HUVECs (upper right) and HMECs (bottom right) are plotted. Bar = 100 µm.
