Rpmi medium

HeLa, HEK293T, U266 cell lines were obtained from ATCC (Rockland, MD). We verified these cell lines via STR profiling (short tandem repeat analysis of DNA). Cell lines that contained knockout or overexpression constructs were not passaged beyond ~ 8 generations (1–1.5 months).
HeLa and HEK293T cells were cultured in in Dulbecco’s Modified Eagle’s medium (Corning, 10–013-CM) with 10% FBS (Peak serum, PS-FB1). U266 cells were cultured in RPMI medium (GE Healthcare Life Sciences, SH30255.01) with 10% FBS.
We generated cells in which TRIM44 was overexpressed (TRIM44[OE]) or knocked down (TRIM44[KD]) via a lentivirus. Control cells were infected with relevant control vectors corresponding to the infecting virus vector (TRIM44[OE-CON] or TRIM44[KD-CON]). Lentivirus were packaged using transfer vector (OHS5898–202620525, or RHS4430–200177847), viral packaging (psPAX2), and viral envelope (pMD2G) in 293FT cells.

THP-1 cells (derived from a male donor) were purchased from ATCC and incubated at 37 °C with 5% CO₂ in RPMI medium (GE Healthcare, Munich, Germany), with the addition of 10% FCS, 1% pyruvate, 1% glutamine, and 1% penicillin/streptomycin (PAA Laboratories, Cölbe, Germany). ETFDH knockdown was induced by transfecting 2 × 10⁶ THP-1 cells with 50 nM small interfering RNAs (siRNAs) (ON-TARGETplus SMART pool, human ETFDH, Thermo Scientific, Karlsruhe, Germany), using Hiperfect (Qiagen, Hilden, Germany). For clustered regulatory interspaced short palindromic repeats (CRISPR)-mediated knockout of TMEM126B, THP-1 cells, stably transduced with a lentiviral vector containing Cas9 (pLentiCas9-Blast; Addgene #52962) and selected with 50 µg/mL blasticidine for 10 days, were again transduced with a lentivirus expressing the guide RNA against TMEM126B (pLentiCRISPRv2 ΔCas9 (derivate of pLentiCRISPRv2; Addgene #52961)). Afterwards, single-cell clones were created. For the experiments, five clones were selected, which showed knockout at the protein level. For the control, a non-target guide RNA (sgC) was used. For the metabolic studies, MDA-MB-231 cells (ATCC) were cultured in Dulbecco’s Modified Eagle’s Medium (DMEM) medium (GE Healthcare), with the addition of 10% fetal calve serum (FCS), 1% glutamine, and 1% penicillin/streptomycin (PAA Laboratories, Cölbe, Germany).

We then next tested weather DCs have an impact on T-cell proliferation. For this purpose, we performed a CFSE assay in order to analyze the effector function of DCs. We isolated DCs from C-HD/OL-HD and mixed them with frozen PBMCs from healthy volunteers by performing MLR [27 (link), 28 (link)]. The allogenic PBMCs were labelled with CFSE (carboxyfluorescein diacetate succinimidyl ester; Molecular Probes, Madrid, Spain) and exposed to mature DCs from HD patients. Allogeneic CFSE-labelled PBMCs (2×10⁵) were co-cultured in 96-well round-bottom plates in the presence of DCs (2×10⁴) collected at the end of the 6-day culture at a 1:10 DCs-to-T-cell ratio. The mixt culture was incubated in RPMI medium (GE Healthcare Life Sciences HyClone Laboratories, Logan, UT, US) supplemented with 10% FBS (Cultek, Madrid, Spain), 100 U ml-¹ of penicillin, 100 μgml^-1 streptomycin and 2mM L-Glutamine at 37° and 5% CO₂ conditions. After 5 days of culture, the cells were labelled with CD3 (Clone HIT3a) APC (Becton-Dickinson Pharmingen) to analyze T-cell proliferation by flow cytometry by FACS CANTO^TMII and analyzed by FACS DIVA software.

Parental THP-1 cells and THP1-XBlue^™-CD14 reporter cells (carrying an NF-κB/AP-1-inducible SEAP reporter construct) were obtained from InvivoGen (USA) and cultured in RPMI medium (GE Healthcare, USA) supplemented with 10% fetal calf serum (FCS, Thermo Scientific, USA), 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM glutamine, and 0.1 M NaHCO3 (all PanEco, Russia) at 37°C with 5% CO₂. 200 μg/ml Zeocin, and 250 μg/ml G418 (both InvivoGen, USA) was included in the culture medium for the THP1-XBlue^™-CD14 reporter cell line.
HEK-Blue-hTLR4, HEK-Blue-hNOD2 and control HEK-Blue-Null2 cells were obtained from Invivogen (USA) and maintained in DMEM medium (GE Healthcare, USA) supplemented with 10% fetal calf serum (Thermo scientific, USA), 50 U/ml penicillin, 50 μg/ml streptomycin, 2 mM glutamine, 0.1 M NaHCO3 (all PanEco, Russia), and 200 μg/ml Zeocin (InvivoGen, USA) at 37°C with 5% CO₂.