Cells were incubated with C2I alone or with C2I + C2IIa or left untreated for control. After 6 h cells were lysed and equal amounts of NKL 22 lysate S-[(1E)-1,2-dichloroethenyl]–L-cysteine proteins incubated with fresh C2I and biotin-labelled NAD+. The biotinylated, i.e. ADPribosylated actin is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins. C. C2IN-C3lim is not taken up into preosteoblastic MC3T3 cells under comparable conditions. Cells were incubated with C2IN-C3lim or with C2IN-C3lim or left untreated for control. After 6 h the cells were lysed and the ADP-ribosylation status of Rho determined as described in A. The biotinylated, i.e. ADPribosylated Rho is shown. Equal amounts of loaded protein were confirmed by Ponceau S staining of the blotted proteins. C2IN-C3lim. Therefore, C2IN-C3lim was used for further experiments. Figure 4A shows the morphology of RAW 264.7 cells cultured in the absence of C2IN-C3lim. Here, numerous multi-nucleated, positively stained osteoclasts were formed during the differentiation of RAW 264.7 cells. In contrast, treatment with C2IN-C3lim reduced the number of multinucleated, TRAP-positive osteoclasts in a concentrationdependent manner and the C3-treated cells formed pronounced cellular protrusions. To investigate the inhibitory effect of C3-treatment on osteoclast-formation in more detail, it was tested whether the time point of C3-application was crucial to inhibit osteoclastformation. To this end C2IN-C3lim was added to RANKLtreated RAW 264.7 cells either from day 0 on, or from day 1 on, or from day 2 on. For control, cells were incubated with RANKL but without C2INC3lim. At day 5 the number of multi-nucleated, TRAP-positive cells was determined. The strongest inhibitory effect on osteoclast-formation was observed when C2IN-C3lim had been added from day 0 on, i.e. in the early stage of osteoclastdifferentiation. Application of C2IN-C3lim from later points in time on did not reduce the number of multi-nucleated, TRAP-positive cells to a comparable degree. Furthermore, a single-pulse incubation with C2INC3lim only at day 0 and subsequent removal of C2IN-C3lim from the cultures by medium change led to a
