Huntingtin depletion impairs Golgi reformation after microtubule disruption. A) HeLa cells were sequentially transfected with scRNA or siRNA-htt and pARIS-mCherry-httQ23/Q100 and finally analyzed by western blot using antibodies that recognize either endogenous and exogenous htt (htt-4C8) or only exogenous htt (HA). Treatment with siRNA-htt (second lane) results in the complete silencing of endogenous htt. Compared to endogenous htt, pARIS-htt displays lower mobility due to fusion with tags. Note that expression levels of pARIS-mCherry-httQ23/Q100 are not modified by siRNA-htt treatment (lanes 4 and 6). α-tubulin is used as a protein loading control. (B) HeLa cells were transfected with scRNA or siRNA-htt, fixed and processed for staining of a Golgi marker (Ctr 433) and α-tubulin. Unlike scRNA-treated cells, cells silenced for endogenous htt display a dispersed Golgi phenotype but an intact MT network. (C) α-tubulin staining before and after nocodazole (NZ) treatment reveals that MT network is entirely reformed 120 min after NZ removal in HeLa cells. (D) A schematic description of the transfection protocol is summarized. (E) HeLa cells stably expressing GFP-mannosidase II were transfected with scRNA or siRNA-htt and treated with NZ for 120 min to allow a complete MT depolymerization. Golgi reformation was monitored 120 min after NZ washout. In scRNA-treated cells the GA becomes again centrally organized. However, cells depleted from endogenous htt still present a dispersed GA at the same time point. Scale bars 10 μm.
Pardo et al. Molecular Brain 2010 3:17 doi:10.1186/1756-6606-3-17