Fig. 5. CHIR99021 neuroprotection in HD patient neurons requires CAST.

a Knock-down efficiency of CAST and calpain I (CAPN1) short hairpin RNA (shRNA) was confirmed by western blotting. b Mitochondrial membrane potential (MMP) was assessed using a TMRM fluorescence probe in control shRNA (shCon) or CAST shRNA (shCAST) expressing cells treated with CHIR99021 (chir) or vehicle (veh) (n = 4). c Cell viability was assessed by MTT assay in shCon- or shCAST-expressing cells treated with CHIR99021 or vehicle (n = 6). d MMP was measured by TMRM staining in control shRNA (shCon)- or shCAPN1-expressing cells treated with CHIR99021 or vehicle (n = 4). e Cell viability was measured by MTT assay in shCon or shCAPN1 cells treated with CHIR99021 or vehicle (n = 8). Mixed striatal neurons were differentiated from iPS cells of patients with HD and normal subjects. Twenty days after neuronal differentiation, neurons were infected with lentivirus expressing GFP-labeled shCon or shCAST. f Knock-down efficiency of shCAST in neurons derived from HD patient iPS cells was confirmed by western blotting. Two days after infection, neurons derived from iPS cells from normal subjects (Nor) and HD patients (HD) were treated with CHIR99021 at 1 μM for 5 consecutive days. g MMP was assessed using a TMRM fluorescence probe (n = 4). Cell viability was measured by MTT assay after brain-derived neurotrophic factor (BDNF) withdrawal for 12 h (n = 4). h Neurons were stained with anti-MAP2 (red)/anti-GAD67 (blue) or anti-β-tubulin (clone Tuj1) (blue)/anti-DARPP-32 (red) to indicate dendritic and axonal morphology, respectively. Scale bar = 30 μm. i Quantification of MAP2⁺ neuronal dendrite length (n = 23 cells/group). j Quantification of Tuj1⁺ neuronal axon length (n = 28 cell/group). All values are reported as mean ± SEM and compared using one-way ANOVA with Tukey’s post hoc test. Data are representative of at least three independent experiments. Exact p values are shown in the figures.