Vimentin affects the mutant Htt inclusion formation in 150Q Neuro2a cells and mediates the effect of Y-27632. A. Immunoblot demonstrating inhibition of vimentin phosphorylation at Ser71 and Ser38 by ROCK inhibitor Y-27632 (20 μM) in Neuro2a cells. B. tNHtt-60Q-EGFP (green) was transfected to Neuro2a cells stably expressing RFP-vimentin (red). Treatment of these cells with 20 μM Y-27632 resulted in filament-like distribution of vimentin and disruption of vimentin cages observed around tNHtt-60Q-EGFP inclusions in the untreated cells. Nuclei were stained with DAPI (blue). Scale bar, 15 μm. C. The effect of Y-286432 on polyQ inclusion formation depends on vimentin level (high vimentin levels enhance inclusion formation). 150Q Neuro2a cells were transfected with vimentin shRNA and 48 hrs later, the cells were induced and treated with 20 μM Y-27632. After 24 hrs, cells were fixed and the inclusion formation was quantified by ArrayScan. *p = 0.00003, ** p = 0.0012, ***p = 0.00003, ****p = 0.0018. D. The effect of vimentin knock-down and Y-27632 on polyQ cytotoxicity. Cells were incubated with PI and the PI-positivity was quantified by ArrayScan. *p = 0.0022, ** p = 0.002, ***p = 0.001, ****p = 0.017. E. The effect of Y-286432 on polyQ inclusion formation is dependent on vimentin phosphorylation (vimentin phosphorylation enhances inclusion formation). 150Q Neuro2a cells were transfected with RFP, WT and phospho-mutant (A2 and E2) forms of RFP-vimentin. Cells were induced and treated with 20 μM Y-27632 for 24 hrs, fixed and analyzed by ArrayScan. *p = 0.00016, **p = 0.0003, ***p = 0.0004, ˆp = 0.0021, ˆˆp = 0.018, #p = 0.04, ##p = 0.022. F. The effect of vimentin over-expression and Y-27632 on polyQ-induced cell death. Dead cells were detached and removed during samples preparation for analysis. Cells that remained attached were counted by ArrayScan. *p = 0.049, **p = 0.008, ˆp = 0.044, ˆˆp = 0.045, #p = 0.032, ##p = 0.034. Bars in C-F represent relative mean values ± s.d. from three independent experiments, with levels under control conditions normalized to a value of 1.