Therefore, C2IN-C3lim was used to investigate the Quisinostat effects mediated by C3-catalyzed Rho-inhibition in differentiating osteoclasts and in already differentiated osteoclasts. By using this fusion toxin, we confirmed earlier results by another group that C3-catalyzed Rho inhibition in already differentiated osteoclasts decreases the resorption activity of these cells. It was reported by various groups that Rho activity regulates the formation of the actin ring in osteoclasts which is a prerequisite for bone resorption by these cells. Moreover, we discovered that application of C2INC3lim to RAW 264.7 cells inhibited their RANKL-induced differentiation into osteoclasts in a time-and concentrationdependent manner which might be a consequence of the inhibited proliferation of C2IN-C3lim-treated RAW 264.7 cells. A weaker inhibitory effect on osteoclast-differentiation was observed when C3bot1 was used instead of C2IN-C3lim while enzymatically inactive C3bot1E174Q had no effect on the morphology of RAW 264.7 cells. Moreover, these results confirmed that C2IN-C3lim is an attractive tool to investigate the specific C3-mediated effects in such cells. The concentration-and Ribocil time-dependent inhibition of osteoclastformation by C2IN-C3lim with the strongest effect after a single-dose of C2IN-C3lim at day 0 or C2IN-C3lim-treatment from day 0 on implies an essential role of Rho in the early phase of osteoclast-differentiation. Although the results imply that a time-dependent Rho-inhibition seems to be crucial for osteoclast-formation, the reason for this effect is not known so far. Interestingly, in contrast to RhoU, the expression of RhoA, which are the selective targets of C3 proteins is not upregulated during RANKL-induced osteoclastogenesis. However, it is not clear whether the merely constant expression of RhoA, over time is related to the strong effect that is exerted by C3 in early osteoclast differentiation. Besides its role as a specific inhibitor to investigate the rol
