Hygromycin b

The construction of the human RORγ expression plasmid was described previously (Karaś et al., 2018 (link)). Expression vectors containing human RORγT and mouse Rory/Roryt cDNA and a control pCMV6-XL5 vector were purchased from OriGene Technologies (Rockville, MD, United States). The reporter vector pGL4.35[luc2P/9XGAL4UAS/Hygro] was purchased from Promega (Madison, WI, United States). The GAL4-DBD RORα and GAL4-DBD RORγ fusion constructs were described previously (Kumar et al., 2010 (link)) and were a kind gift from Prof. Patrick Griffin. Hygromycin B (P06-08020) was purchased from PAN Biotech GmbH. Digoxin (D6003, purity ≥ 95.0%) was purchased from Sigma-Aldrich (Saint Louis, MO, United States).

HeLa cells were cultured in DMEM supplemented with 10% FBS in the presence of penicillin/streptomycin (pen/strep). Stable inducible p97‐GFP HeLa FRT cells were cultured in DMEM supplemented with 10% tetracycline‐free FBS in the presence of pen/strep with 250 μg/ml Hygromycin B (PAN‐Biotech) and 15 μg/ml Blasticidin (ThermoFisher Scientific). Stable mCherry‐Gal3 HeLa cells were cultured in DMEM supplemented with 10% FBS in the presence of pen/strep with 500 μg/ml G418 (VWR). Cells were transfected with plasmids using Lipofectamine 2000 (ThermoFisher Scientific) or with 10 nM siRNA (or 20 nM for the screen) using RNAiMAx (ThermoFisher Scientific) according to the manufacturer's instructions. Transfected cells were analyzed after 24 h (plasmids) or 48, 60 or 72 h, as indicated (siRNA). For rescue experiments, cells were transfected with siRNAs for a total of 72 h. Medium was changed after 24 h, and cells were transfected with plasmids for another 48 h.

Cells of the human neuroblastoma SH-SY5Y cell line were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM), containing 10% fetal calf serum (FCS, GE Healthcare Life Sciences, Chalfont St. Giles, United Kingdom) and 0.1% non-essential amino acid solution (MEM). For the African green monkey fibroblast-like cell line COS7, culture medium contained DMEM and 10% FCS. For the stably transfected cell lines SH-SY5Y/COS7 APP⁶⁹⁵, which overexpress the major neuronal APP isoform in humans, APP⁶⁹⁵, and for stably transfected SH-SY5Y C99 cell line^{60 (link)}, which overexpress the β-secretase cleavage product, β-CTF, 400 µg/mL hygromycin B (PAN-Biotech, Aidenbach, Germany) were added to the media. For the murine neuroblastoma cell line (N2a), culture medium consisted of DMEM, 10% FCS, 0.1 mM MEM, 0.1 mg/mL Streptomycin, 100 U/ml Penicillin, 2 mM L-Glutamine and 1 mM sodium pyruvate. For N2a cells with IDE knock-down, promoted by SureSilencing shRNA plasmids (Qiagen, Hilden, Germany) regarding to manufacturer’s protocol and^{61 (link)}, 400 µg/mL hygromycin B were added to the media. Knock-down efficiency is shown in Supplemental Fig. S4.

GFP-TAFs fusion cell lines used in this study were described in ref. ²⁶ . Briefly, the coding sequences for the human TFIID subunits (TAF1, TAF2, TAF3, TAF4, TAF5, TAF6, TAF7, TAF8, TAF9, TAF10, TAF11, TAF12, TAF13 and TBP) were obtained by PCR using the appropriate cDNA clone and gene-specific primers flanked by attB sites followed by BP-mediated GATEWAY recombination into pDONR221, according to the manufacturer’s instructions (Invitrogen). The cloned sequence was verified by sequencing and it was transferred to the pcDNA5-FRT-TO-N-GFP Gateway destination vector by LR recombination, according to the manufacturer’s protocol (Invitrogen). HeLa Flp-In/T-REx cells, which contain a single FRT site and express the Tet repressor^{25 (link)}, were grown in DMEM, 4.5 g L^–1 glucose (Gibco), supplemented with 10% vol/vol fetal calf serum (Gibco). All the GFP-fusion destination vectors were co-transfected with a pOG44 plasmid that encodes the Flp recombinase into HeLa Flp-In/T-REx cells using polyethyleneimine (PEI) to generate stable doxycycline-inducible expression cell lines. Recombined cells were selected with 5 μg ml^–1 blasticidin S (InvivoGen) and 250 μg ml^–1 hygromycin B (Roche Diagnostics) 48 h after PEI transfection. Cells were maintained in DMEM supplemented with 10% Tet-free fetal calf serum (Pan Biotech, P30-3602), blasticidin S, hygromycin B and penicillin–streptomycin.

SH-SY5Y wildtype (wt) cells were cultivated in Dulbecco’s Modified Eagle’s Medium (DMEM) containing 10% fetal calf serum (FCS) (PAN Biotech, Aidenbach, Germany). For SH-SY5Y APP (overexpressing the human APP695 isoform) and SH-SY5Y C99 cells (overexpressing the β-cleaved C-terminal fragment) [78 (link)], Hygromycin B (PAN Biotech) was supplemented in a final concentration of 0.3 mg/mL.
Neuro 2a (N2a) cells were cultivated in DMEM comprising 10% FCS, 1% penicillin/streptomycin solution, 2 mM l-glutamine, 0.1 mM MEM and 1 mM sodium-pyruvate.
Twelve hours before incubation, FCS in cell culture medium was reduced to 0.1%.
Tocopherol and tocotrienol (dissolved in ethanol) were incubated in a concentration of 10 µM in 0.1% FCS/DMEM for 24 h (8 + 16 h); controls were treated with the appropriate ethanol concentration (1%). Depending on subsequent experiments, cells were either lysed chemically in lysis buffer (0.1% NP-40, 0.1% Triton-X-100, 10 mM Tris, 2 mM EDTA) with or without protease inhibitor (Roche Diagnostics, Mannheim, Germany) or homogenized mechanically using a Minilys homogenizer (Peqlab, Erlangen, Germany).

Hela and A549 cells were grown in DMEM medium (PAN Biotech, Aidenbach, Germany) supplemented with 10% fetal bovine serum (FBS, GE Healthcare) and 100 µg/ml penicillin/streptomycin (Pen/Strep, PAN Biotech). 116 cells were grown in MEM eagle medium (PAN Biotech) supplemented with 10% FBS, 100 µg/ml penicillin/streptomycin and 100 mg/ml hygromycin B (PAN Biotech). Primary human skeletal myoblasts (HSKM) were grown in DMEM supplemented with 20% FBS, 1% gentamycin and 1x Glutamax (Thermo Fischer Scientific). All cells were cultured at 37 °C and 5% CO₂ in a standard tissue culture incubator.