Figure 4

LYHY causes apoptosis with no change in TER. A. Eph4 cells were grown on glass slides and treated with LYHY or control LYQY peptide for 4, 8, and 12 hours. Cells were TUNEL stained and counted (see Additional File 1A). B. Eph4 cells were peptide treated and stained live with sulphorhodamine-DEVD-fluoromethyl ketone for activated caspase 3 (C-3) and with carboxyfluorescein-LETD-fluoromethyl ketone for caspase 8 (C-8) (see Additional File 1B), fixed, photographed and counted. C. Primary cultures from the mammary gland of a control mouse and an occludin null mouse were treated with the LYHY peptide and stained with an antibody for cleaved caspase 3. Control is no peptide. **p < 0.01 compared to LHYH treated Occ⁺⁺ cells, middle bar. D. Mature Eph4 monolayers on glass slides were pre-treated for 2 hours with a peptide inhibitor of caspase 8/10 or caspase 9, or solvent only (Not inhibited). Cells were then treated with LYHY peptide for 2 hours and the number of cells showing activated caspase 3 counted. E. Eph4 cells were grown on filters until the TER was ~1400 (Ohms•cm2), then treated with solvent, LYHY, or control LYQY peptide at 350 μM in both top and bottom chambers. Peptides were replenished daily, after recordings. Asterisk indicates a statistically significant fall in TER at 72 hours (p < .05) relative to control peptide. Results of three wells averaged.