ch-4C5 inhibis cancer cell invasion in <i>vitro</i>.

<p><b>A</b>. Wound healing assay. Photographs represent phase-contrast images obtained at zero time (left panel) and at 24 h (right panel) after scratch formation, showing MDA-MB-453 cell migration either in control cultures or cultures including anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5. Scale bar: 200 µm. <b>B</b>. Quantitative effect of antibodies on the closure of the wound. Addition of 200 µg/ml of anti-HSP90α and mAb 4C5 in the culture medium resulted in a 48% and 55% reduction of wound closure, respectively when compared to control cultures that were considered as resulting in 100% wound closure. Addition of 200 µg/ml rec-4C5 and ch-4C5 in the culture medium resulted in a 46% and 46% inhibition of wound closure, respectively. Statistical significance of differences was assessed by Student's t test. The presence of anti-HSP90α, mAb 4C5, rec-4C5 or ch-4C5 had a statistically significant effect on the wound closure (p<0.01 in each case). <b>C</b>. Phase-contrast images obtained at zero time (left panel) and at 24 h after scratch formation (right panel), showing B16 F10 melanoma cell invasion in a wound healing assay in the presence of 200 µg/ml of ch-4C5. Scale bar: 200 µm. <b>D</b>. Quantitative effect of increasing concentrations of ch-4C5 on the invasion level of B16 F10 melanoma cells. Presence of 50 µg/ml ch-4C5 resulted in 15% inhibition of invasion, while addition of 100 µg/ml and 200 µg/ml ch 4C5 resulted in 21% and 43% inhibition of migration, respectively when compared to control cultures that were considered as resulting in 100% wound closure. <b>E</b>. Visualization of dead cells using trypan blue dye. In ch-4C5 treated cultures, the cell death incidence is similar to that observed in the control cultures. In contrast, in the cultures treated with the anti-HSP90α antibody, a much greater number of cells are stained with Trypan blue, indicating an increased incidence of cell death Scale bar, 30 µm. <b>F</b>. Control and ch-4C5 treated cells were fixed permeabilized and stained with fluorescently labelled phalloidin. Scale bar 40 µm <b>G</b>. Higher magnification showing phalloidin staining (F-actin). Ch-4C5 effectively blocks spreading of lamellipodia. Scale bar: 16 µm.