Dopamine/D2 Receptor and HD Figure 4. D2-R mediated neuritic retraction and growth cone collapse is associated with ROCK-II activation in striatal neurons. A) Quantification of mean neuritic length in neurons transfected with Htt (white bars) or ExpHtt (black bars) was determined from MAP-2 staining. Transfected neurons were pre-treated with ROCK pharmacological inhibitors Y27632 (10 mM) or Hydroxyfasudil (10 mM) 30 minutes prior to and during quinpirole treatment (10 mM) (14 hours). Data are expressed as mean6SEM from 3 independent experiments (100 transfected neurons per condition and per experiment) (***P,0.001). B) Striatal extracts from adult mouse (striatal extracts), striatal neurons in culture (striatal cultures) and HEK293 cells extracts (HEK cells) were processed for Western-blot with specific antibodies against ROCK-I (left) and ROCK-II (right). Similar amounts of proteins were loaded in each case. b-tubulin was used for control loading. Note the high expression level of ROCK-II in both striatal extracts and culture when compared to ROCK-I expression. C) Control siRNA (50 nM) or siRNA targeting ROCK-II (50 nM) were transfected in primary striatal neurons for 72 hours. Extracts from siRNA-transfected or non transfected (control) neurons were processed for Western-blot with specific antibodies against ROCK-II and b-tubulin. Note the knock-down of ROCK-II expression. D) Quantification of mean neuritic length in neurons transfected with Htt (white bars) or ExpHtt (black bars) after quinpirole treatment (10 mM) alone or in the presence of control siRNA (50 nM) or ROCK-II siRNA (50 nM). Data are expressed as mean6SEM from 3 independent experiments (100 transfected neurons per condition and per experiment). (***P,0.005, **P,0.05). E) Quantification of growth cone collapse in neurons transfected with Htt (white bars) or ExpHtt (black bars) after quinpirole treatment (10 mM) for 14 hours alone or in the presence of ROCK inhibitors (10 mM) or ROCK-II siRNA (50 nM). Data are expressed as mean6SEM from 3 independent experiments (100 transfected neurons per condition and per experiment). In these experiments, neurons transfected with control siRNA showed no significant difference with non transfected neurons (control) alone (***P,0.001). doi:10.1371/journal.pone.0008287.g004 PLoS ONE | www.plosone.org 5 December 2009 | Volume 4 | Issue 12 | e8287