FIGURE 3:
Farnesyltransferase inhibition or prevention of Spindly farnesylation impairs kinetochore recruitment of dynein and its cofactor dynactin. (A–D) HeLa cells immunostained for dynein IC (DIC) and the dynactin subunit p150Glued in cells treated with FTI-277 or DMSO (A, B) and in cells expressing wild-type (WT) and mutant (C602S) MycGFP::Spindly after RNAi-mediated depletion of endogenous Spindly (C, D). Cells were incubated in 1 μM nocodazole for 4 h to maximize the accumulation of the proteins at kinetochores and costained with ACAs. Scale bars, 5 μm. (E, F) Quantification of dynein and dynactin levels at kinetochores in the conditions shown in A–D, as well as in the Spindly mutant C602S after treatment with FTI-277, using immunofluorescence intensity measurements. Each condition represents a total of 100 kinetochore measurements from 20 different cells. Error bars represent the SEM with a 95% confidence interval. The t test was used to determine statistical significance (***p < 0.0001; ns, not significant).