Fig. 2. Melanoma cell rounding, de-adhesion and membrane blebbing induced by clustered ephrin-A5 Fc is inhibited by soluble, monomeric ephrin-A5. (A) LiBr, AO9 and AO2 melanoma cells on fibronectin-coated glass coverslips were stimulated with 10 nM clustered ephrin-A5 Fc. Soluble, monomeric ephrin-A5 was added as inhibitor (+ inhibitor) to a parallel LiBr culture at 100-fold molar excess. Selected pictures of representative microscopic images taken during a 50 minute time course are shown. Arrowheads denote cells that retract their processes and round up during stimulation. Bar, 20 µm. A supplemental video of the AO9 time course is available at: http://www.ludwig.edu.au/addinfo/Figure2_AO9.mpg. (B) LiBr melanoma cells on fibronectin-coated glass coverslips before (-) or after (+) 10 minute ephrin-A5 stimulation were fixed, permeabilised and stained with rhodamine-phalloidin to visualise polymerised actin. Bar, 20 µm. (C) Melanoma cells on fibronectin-coated 96-well plates were treated as decribed in (A) and at indicated times cells were fixed and stained with crystal violet. Values represent mean A₅₅₀ absorbances from quadruplicate wells ±s.d.: , LiBr cells; , AO9 cells; , LiBr cells with inhibitor; [UNK], AO2 cells. (D) Ephrin-A5 treatment does not impair cell viability in LiBr or EphA3 293T cells. Cultures of LiBr (grey bars) or EphA3 293T (black bars) cells in 24-well plates were treated with preclustered human IgG (control) or ephrin-A5 for 2 hours at 37°C, detached by EDTA treatment and cell viability in quadruplicate samples determined by trypan blue exclusion assay. To induce apoptosis, cells in parallel wells were treated for 4 hours with 1 µM staurosporin (St.sp.) or 0.5 mM H₂O₂. Control cells in parallel wells were treated for 30 minutes with the caspase inhibitor zVAD-fmk (0.1 mM) before addition of the apoptotic reagent. (E) Ephrin-A5 stimulation of LiBr cells does not result in increased numbers of apoptotic cells. LiBr monolayer cultures were treated for 2 hours at 37°C with (a), 1.5 µg/ml or (b), 7.5 µg/ml pre-clustered ephrin-A5 Fc; (c) 7.5 µg/ml preclustered control IgG; and (d) 1 µM staurosporin. Cells in the supernatant and detached by trypsin/versene treatment were combined, washed in Annexin V binding buffer and analysed for binding of Annexin V to the outer plasma membrane (apoptosis) and propidium iodide (PI) uptake (necrosis) by flow cytometry. Viable cells (lower left quadrant) are negative for Annexin V and PI, apoptotic cells (upper left quadrant) are positive for Annexin V, necrotic cells (upper right quadrant) are positive for Annexin V and PI.