(a)
(b)

Figure 3: The impact of CD44v6 and Tspan8 on TEX binding and uptake. (a) Dio-labeled A818.4-wt, -CD44v6kd and -Tspan8kd TEX were incubated with A818.4 wt, -CD44v6kd, and -Tspan8kd cells for 3h and 6h at 37°C. After washing, uptake of Dio-labeled dye was evaluated by flow cytometry; mean ± SD of dye-labeled cells in 3 experiments is shown; (b) uptake of CIC-TEX (holoclone-derived) from GFP transfected A818.4 cells (GFP-TEX) was evaluated by confocal microscopy after counterstaining with anti-CD44v6 or anti-Tspan8. The experiment was repeated 3 times. Representative examples are shown (scale bar: 10μm). A818.4-wt TEX are taken up by -wt, -CD44v6kd, and -Tspan8kd cells; instead A818.4-CD44v6kd and -Tspan8kd TEX are poorly taken up, indicating that CD44v6kd and Tspan8kd cells express ligands for A818.4-wt TEX binding, but CD44v6kd- and Tspan8kd-TEX miss receptors/receptor complexes. Uptaken TEX preferentially colocalize with Tspan8.