Fig. 4. (A) Inhibition of PKC dose-dependently reduced CytD-induced adhesion. KG1 cells were pre-treated for 10 minutes at 37°C with increasing concentrations of either chelerythrine chloride (black bars) or myristoylated PKC inhibitor (white bars) in combination with CytD (2.5 µg/ml). Both inhibitors of PKC significantly reduce CytD-induced adhesion to immobilized ALCAM. (B) PKC requirement is not restricted to CytD but applies to LatA as well. KG1 cells were pre-treated with chelerythrine chloride (Chel, 5 µM) or myristoylated PKC inhibitor (Myr, 100 µM) for 10 minutes at 37°C in combination with either CytD (2.5 µg/ml, black bars) or LatA (5 µg/ml, gray bars). Induction of adhesion by both cytoskeleton-disrupting drugs is equally inhibited by both chelerythrine chloride and myristoylated PKC inhibitor, demonstrating that the requirement for PKC is not restricted to the mode of action of CytD in itself. Similar observations were made for K562-ALCAM (not shown). (C) Analysis of the distribution of ALCAM and actin by CLSM. KG1 cells were pre-treated without (a) or with CytD (2.5 µg/ml, b), or with a combination of CytD and chelerythine chloride (Chel, 5 µM, c). Cells were stained with mAb AZN-L50 (green) and Texas Red-X phalloidin (red). For each preparation the same instrument settings were used. The scale bar represents 10 µm. Similar results were obtained with K562-ALCAM cells (not shown). (D) PKC requirement is not restricted to homotypic ALCAM-mediated adhesion induced by cytoskeleton-disrupting agents, but also applies to heterotypic ALCAM-CD6 adhesion. KG1 cells were pre-treated with PKC inhibitors as described in B, in the presence or absence of CytD (2.5 µg/ml). Cells were allowed to adhere to a plate coated with 250 ng/ml of CD6-Fc (white bars) or ALCAM-Fc (black bars) for 45 minutes at 37°C. Both spontaneous and CytD-induced ALCAM-CD6 adhesion are inhibite d by chelerithrine chloride and myristoylated PKC inhibitor. Similar observations were made for K562-ALCAM cells (not shown).