Figure 2.

Effect of TG2 inhibition on endothelial tubule formation in fibroblasts and EC co-cultures. (a) After incubating the V2a AngioKit co-culture for 24 h, V2a Growth medium was introduced (day 1) in the absence or presence of either the irreversible inhibitors Z-DON, R294 or R283 at the concentrations shown, and replaced in fresh medium every other day for 12 days. Controls contained either complete growth media alone (untreated), or the respective inhibitor vehicle (DMSO, 0.01%). Suramin or VEGF was used for the negative and positive control, respectively. Cells were fixed in ethanol at day 12 and stained for CD31 antigen using an anti-mouse IgG secondary AP-conjugated antibody and visualised as described in the Materials and Methods. The tubule like structures were analysed by the TCS Cellworks AngioSys Image Analysis Software (ZHA-1800) (Supplementary Table S1), as described in the Materials and Methods. (b) The presence of TG2 in human fibroblasts and HUVECs. Western blotting was performed to detect the presence of TG2 in fibroblasts and HUVECs, separately. α-Tubulin was used as the equal loading control. (c) Co-culture of HUVEC and TG2−/− MEF. HUVECs were induced to undergo microtubule formation in the presence of TG2−/− fibroblasts using the growth media from the angiogenesis V2a Kit. Shown is the appearance of endothelial cell microtubules at day 14 revealed by immunostaining for CD31 antigen using anti-mouse IgG secondary alkaline phosphatase (AP)-conjugated antibody and visualised as described in the Materials and Methods.(d) Effect of different TG2-specific antibodies on endothelial cell tubule formation in the co-culture assay. Culture medium was supplemented with either the mouse monoclonal TG2 activity neutralising antibody (D11D12, 0.1 μg/ml), the commercial anti-TG2 antibodies Cub7402 (0.5 μg/ml) or TG100 (0.5 μg/ml) added to the co-culture system from day 1 (24 h after seeding). Controls consisted of either untreated cultures or isotype-matched IgG. After 12 days, the tubule formation was analysed as described in (a) (see Supplementary Table S1). (e) The effect of the antibodies D11D12, Cub7402 and TG100 on the extracellular crosslinking activity of TG2 in the co-culture angiogenesis assay. Cells at day 9 were incubated with various concentrations of the antibodies or isotype-matched control for 1 h at 37 °C in a 96-well microplate. Following this pre-incubation period, biotinylated cadaverine incorporation was measured as described in the Materials and Methods. Data show mean value±S.D. after background correction (n=3) for assays that contained 10 mM EDTA