T rex 293 cells

The ability of DNAJB6 to prevent aggregation of polyQ-huntingtin was tested using filter-trap assay as described earlier [1 (link),5 (link),9 (link)] with minor modifications. In brief, T-REx 293 cells (Invitrogen) were grown at 37 °C, 5% CO₂, in DMEM supplemented with 10% fetal bovine serum, GlutaMax, penicillin/streptomycin, and blasticidin S. The cells were transfected with GFP-tagged 120Q-HTT and inducible V5-tagged DNAJB6-constructs, expression of DNAJB6 induced after 5 h (or 4 h in experiments involving p.H31Q constructs) and the cells harvested 48 h post transfection. The soluble fraction of 120Q-HTT was measured using western blotting and the aggregates trapped in a 0.2 μm cellulose acetate filter (Whatman GmbH). The membranes and filters were stained for GFP (A-11122, Invitrogen) and V5 (R960-25, Invitrogen), scanned on an Odyssey scanner (LI-COR) and quantified using ImageStudio v. 3.1.4 (LI-COR) or ImageJ. The aggregation score was calculated from the intensities of aggregated and soluble GFP-HTT as (aggregated/soluble)_induced/(aggregated/soluble)_uninduced and statistical significance calculated using a two-tailed Mann–Whitney U test without multiple testing correction.

All cell lines were grown in medium supplemented with 10% foetal bovine serum (FBS, Pan Biotech), 100 units ml^-1 of penicillin and 100 μg ml^-1 of streptomycin (Gibco), at 37 °C in a humid atmosphere containing 5% CO₂. Human embryo kidney (HEK) 293T cells (ATCC, CRL-11268) and monkey kidney BS-C-1 (ATCC, CCL-26) and CV-1 (ATCC, CCL-70) cells were grown in Dulbecco’s modified Eagle’s medium (DMEM, Gibco). Rabbit kidney RK13 cells (ATCC, CCL-37) were grown in minimum essential medium (MEM, Gibco) and human cervix HeLa cells (ATCC, CCL-2), in MEM supplemented with non-essential amino acids (Gibco). T-REx-293 cells (Invitrogen) were grown in DMEM supplemented with blasticidin (10 μg ml^-1, InvivoGen), whilst the growth medium of T-REx-293-derived cell lines stably transfected with pcDNA4/TO-based plasmids was further supplemented with zeocin (100 μg ml^-1, Gibco). The absence of mycoplasma contamination in the cell cultures was tested routinely with MycoAlert detection kit (Lonza), following the manufacturer’s recommendations.

T-REx™-293 cells (Invitrogen R71007) were transfected with the plasmid pCDNA5-TO-h-SLC1A5 (Invitrogen V1033-20). The cloning sites consisted of Not1 and Xba; insert OriGene # SC116600. Stably transfected clones were selected via hygromycin (150 µg/ml).

T-REx-293 cells (Invitrogen), which constitutively expresses the Tet repressor (TetR), were transfected with pcDNA4/TO-coF14-TAP plasmid, which encodes F14 under the control of the HCMV immediate early promoter and two tetracycline operator (TetO₂) sites. Transfected cells were selected in the presence of blasticidin (10 μg ml^-1) and zeocin (100 μg ml^-1) and clonal cell lines were obtained by limiting dilution. Expression of protein F14 within these clones was analysed by immunoblotting and flow cytometry with anti-FLAG antibodies. T-REx-293-EV, T-REx-293-B14 and T-REx-293-C6 cell lines were described elsewhere^{21 (link)}.