Figure 5.

The effect of TG2 inhibition of HUVEC morphology and FN deposition. (a) Representative images (n=3) from monolayer HUVECs treated with 100 μM R294 or vehicle for 48 h and the actin cytoskeleton (green) and FAs (vinculin, red, as pointed out by the arrow heads) staining was performed and visualised as described in the Materials and Methods. Nuclear counterstaining of cells used 4, 6-diamidino-2-phenylindole (DAPI; blue). (b) Cytoskeletal organisation of HUVEC-TG2wt and HUVEC-TG2kd. Morphological comparison of the spreading ability of HUVEC-TG2wt and HUVEC-TG2kd, when assessed for actin stress fibre formation. HUVECs expressing the viral constructs (GFP, green colour) were plated on FN-coated cover slips in the presence of EGM complemented medium. TRITC-labelled phalloidin (red) was used to stain actin fibres and then examined by confocal microscopy as described in the Materials and Methods. (c) The effect of TG2 on ECM FN deposition by HUVECs. Representative image from HUVECs treated with 100 μM TG2 inhibitor R294 or vehicle for 48 h before immunostaining for extracellular FN as described in the Materials and Methods. Cells were counter stained with DAPI. The ECM FN was visualised using fluorescence microscopy as described in the Materials and Methods. (d) The inhibition of biotin-labelled FN deposition by R294 in HUVECs. Biotin-labelled FN 50 nM was added into HUVEC mono-cell layer as described in the Materials and Methods with 100 μM R294 or with the vehicle control. After a 48-h incubation period, the presence of biotin-labelled FN was detected using Cy5-conjugated Strep-Avidin and the fluorescence signal was visualised via confocal microscopy. Bar, 25 μm