Smartpool

MUTZ-LCs and primary LCs were silenced by electroporation with Neon Transfection System (ThermoFischer Scientific). The siRNA were specific for langerin (10 μM siRNA, M-013059-01, SMARTpool; Dharmacon), siRNA Syndecan 4 (10 μM siRNA, M-003706-01-0005, SMARTpool; Dharmacon) or non-targeting siRNA (D-001206-13, SMARTpool; Dharmacon) as control. Silencing of the targets was verified by real-time PCR, flow cytometry. Cells were used for experiment 72 h after silencing. Silencing of the targets was verified by real-time PCR or flow cytometry.

siRNAs were transfected using a standard calcium phosphate transfection protocol (Lossaint et al., 2013 (link)). siRNAs used were Negative Control siRNA (1027310, QIAGEN), human BRCA2 (ON-TARGET plus SMARTpool, L-003462-00, Dharmacon), human BRCA1 (5′-GGAACCUGUCUCCACAAAG-3′), human ERCC4 (ON-TARGET plus SMARTpool, L-019946-00-0005, Dharmacon), and human PAF1 (ON-TARGET plus SMARTpool, M-020349-01, Dharmacon).

This protocol was adapted from Migneault et al. [18 (link)]. Cells were plated onto 6-well plates at 2500 cells per cm². After 72 h, cells were transfected with ON-TARGETplus nontargeting siRNA #3 – SMARTpool, ON-TARGETplus Human CAV1 siRNA (90 nM) – SMARTpool, ON-TARGETplus Human PCSK5 siRNA (90 nM) – SMARTpool (Dharmacon, Lafayette, CO, USA) using Magnet Assisted Transfection (MATra) (IBA Lifesciences, Göttingen, Germany) according to the manufacturer’s instructions. Briefly, MATra-si Reagent was added to siRNA at a ratio of 1 µL:1 µg in Opti-MEM medium (Gibco) and incubated for 25 min at room temperature. The siRNA-bead mixture was added to the supernatant (Opti-MEM) of each well. Then, the plate was placed on a magnetic plate for 15 min, and the medium was changed after 30 min. After 48 h, the cells were processed according to the assay performed.

Adherent cells were seeded at a density of 3×10⁵ per well and transfected with Dharmacon Smartpool targeting human Vps26A, Vps35 or a non-targeting control siRNA using Oligofectamine based on the method of Seaman [18] (link). Unless otherwise stated, 200 nM of siRNA was used. Twenty-four hours later the cells were reseeded and the following day an identical second transfection was performed. Forty-eight hours later the cells were harvested and Vps26 and Vps35 knockdown were determined by western blotting. HeLa TZM-bl cells were infected with pNL4.3 or pNL4.3-Δ144 virus at an MOI of 0.1 immediately after the second knockdown. Excess virus was removed by washing and cells incubated at 37°C for 48 h prior to analysis. RNAi knockdown of Vps26 in T cells was performed by generating 4 individual oligonucleotide hairpins based on the Dharmacon Smartpool target sequences. shRNA hairpins were cloned individually into the HIV-1 vector pCSRQ, a modified version of pSIREN RetroQ [59] (link) (a gift from G. Towers, UCL) and co-transfected into 293T cells with the VSV-G envelope plasmid. Virions were used to infect T cells and stable cell lines selected with puromycin.

SH-SY5Y cells were maintained in DMEM/F12 supplemented with 10% heat-inactivated FBS, 1% NEAA (Thermo Fisher Scientific (Waltham, MA, USA), #1140-035), and 1% penicillin/streptomycin. For siRNA transfection, cells were seeded 24 h prior to transfecting three consecutive days before harvest and three days after the first transfection (representative blot Miro1 and Tom20). The protocol was modified by transfecting the cells once and harvesting them three days later. Transfection with non-targeting siRNA (Dharmacon (Lafayette, CO, USA) SMART Pool; #D-001206-14-05) and RHOT1 siRNA (Dharmacon (Lafayette, CO, USA) SMART Pool, #M-010365-01-0005) was performed using 25 ng siRNA with 2 µL transfection reagent (Dharmacon (Lafayette, CO, USA), #T-2001-01) per well, following the manufacturer’s instructions.