Neuronal loss and apoptosis measurements
Patient iPS-derived neurons exhibit HD-dependent phenotypes including elevated caspase-3 signals and neuronal loss upon BDNF removal. Fluorescent dye was used to measure the apoptosis phenotype, and Immunostaining of Tuj1, a neuronal-specific-tubulin that capture neuronal morphology, was used to measure neuronal shrinkage and loss phenotype. The STHdh cells exhibit HD-dependent caspase 3 activity upon stress, such as serum removal. For compound treatment in the cells, the compounds were diluted in DMSO to 1X concentrations and added to the cells at least one day after plating (for iPS-derived neurons, the compounds were added one week after plating).The cells were stressed (BDNF removal for iPS-derived neurons) 1 day after compound treatment, and tested at the indicated time points. For caspase-3 activity detection, NucView 488 caspase-3 dye (Biotium, #30029) was used for staining as an indicator for apoptosis. For assaying the neuronal loss, Tuj1, a neuronal specific tubulin was stained to show neuronal morphology. Images were taken by Carl Zeiss microscopes and analyzed blindly by ImageJ for caspase-3 and Tuj1 (Covance, #MMS-435P) quantifications.
Western-blot analysis
Heala cells were plated at 75% confluency 24 wells plate. 16hr after plating the cells, treatments were carried out as indicated concentrations for 24hr. Then cells were lysed on ice in 6XSDS Sample buffer (62.5 mM Tris-HCl pH 6.8, 5% β-mercaptoethanol, 10% glycerol and 0.01% bromophenol blue) for 30 min in presence of protease inhibitors (Roche Diagnostics). Proteins were resolved by SDS-PAGE and transferred onto a NC membrane (Millipore) by semi-dry Western- blot. The membrane was blocked with 5% milk in PBST (1XPBS, 0.5% Tween20) and incubated with the indicated primary antibody in 5% BSA in PBST overnight at 4℃. The membrane was then washed three times using PBST and incubated with the HRP-conjugated secondary antibody (Jackson ImmunoResearch) diluted 1:10000 in 5% BSA with PBST for 1 h at RT, washed again three times (5 min each) with PBST, and incubated with ECL reagent mixture (Millipore). Chemiluminescence signals were detected using the ChemiScope 3400 Mini (Qinxiang).