Figure 2. Dose response and reversibiltiy analysis of centrosomal cohesion deficits and Rab10 phosphorylation in control and G2019S LRRK2 LCLs.

(A) The centrosome phenotype was quantified from five distinct control and five G2019S LRRK2 LCL lines, in either the presence or absence of the indicated concentrations of MLi2 for 2 h. In addition, cells were treated with 50 nM MLi2 for 2 h, followed by incubation in medium without MLi2 for an additional 2 h (washout) before quantification. Bars represent mean ± s.e.m.; * P < 0.05; ** P < 0.01; *** P < 0.005; **** P < 0.001 (one-way ANOVA with Tukey's post-hoc test). (B) Quantification of the percentage of cells displaying duplicated centrosomes, a phenotype mainly reflecting cells in G2 phase, from a total of 100 cells per line, for each of the five control and five G2019S LRRK2 lines. (C) Control LCL was incubated in either the presence or absence of the indicated concentrations of MLi2 for 2 h as indicated, or treated with 50 nM MLi2 for 2 h followed by incubation in medium without inhibitor for an additional 2 h (washout), and extracts analyzed for LRRK2 Ser935, LRRK2, Rab10 Thr73, Rab10 or tubulin as loading control. Membranes were developed using the Odyssey CLx scan Western blot imaging system, and antibodies multiplexed as described in Materials and methods. (D) Same as (C), but performed with G2019S LRRK2 LCL. Similar results were obtained in two independent experiments.