Figure 2. BE(2)C cells expressing variant forms of torsinA presented decreased Gluc secretion and neurite outgrowth as compared with cell expressing torsinAwt.

(A) BE(2)C cells were transduced with the lentiviral vectors encoding torsinAwt, torsinAΔE, torsinAR288Q or torsinAF205I. Forty-eight hr later cells were immunostained for torsinA, PDI, and β-tubulin. (B) BE(2)C cells non-transduced or transduced with vectors expressing wt or torsinA variants were lysed and 30 µg of each sample resolved by SDS PAGE. A representative western-blot shows equal levels of torsinA in all transduced cells. (C) BE(2)C cells expressing torsinA variants were transduced with lentiviral vectors encoding Gluc-IRES-cerulean and Fluc-IRES-mCherry. The cells were replated 48 hr after infection at 6 × 10⁴ cells per well in 96-well plates. The Gluc activity in the medium was determined as RLU at 24 hr after replating the cells. (D) BE(2)C stable cell lines expressing torsinA forms: torsinAwt torsinAΔE, torsinAR288Q, and torsinAF205I were grown for 3 days in presence of blebbistatin and all-trans retinoic acid and visualized by immunocytochemistry using a polyclonal torsinA antibody TA2 (red), a monoclonal antibody specific for PDI (green), and monoclonal antibody β-tubulin-Alexa Fluor 647 (blue). (E) Measurement of neurite length after treatment with blebbistatin and retinoic acid for 3 days in uninfected BE(2)C cells and cells infected with lentiviral vectors encoding torsinAwt and torsinA variants. A two-fold increase corresponds to growth of about 40 µm. The experiments were repeated three times in quadruplicates for each cell line (ANOVA post-hoc Dunnett's t-test ***p<0.001; n=3).