Penicillin

Cells were cultured in Express Five medium (Invitrogen) supplemented with glutamine, penicillin, and streptomycin (Wisent) at 25 °C. Transfections with plasmids were performed using X-tremeGENE HP DNA Transfection Reagent (Roche) following the manufacturer’s protocol. Stable cell lines were selected by adding 20 ug/ml blasticidin into the cell culture after transient transfections of plasmids of interest. While inducible pMT-based vectors contain the gene coding for blasticidin resistance, pAc5-based vectors were co-transfected with pCoBlast to confer resistance to blasticidin to the cells. 300 μM CuSO₄ were added into the medium to induce expression of the plasmids containing pMT promoter, at least 16 hr before use. For RNA interference, dsRNAs were generated from PCR amplicons using a Ribomax Kit (Promega). RNAi non-target (NT) was generated against the sequence of the procaryotic kanamycin resistance gene. 1×10⁶ cells were plated in a six-well plate and treated with 1 ml of medium containing 20 μg of indicated dsRNA for 4 d (rescue experiments). Cells were then harvested and analyzed by immunoblotting, immunofluorescence, or live-cell imaging.

After obtaining informed, signed consent from healthy participants, blood was collected and approved by the Concordia University Human Research Ethics Committee, following the Declaration of Helsinki guidelines (certificate 30009292). Participants’ health status was verified through self-reporting during a semi-structured interview. Individuals under 18, those with certain medical conditions, or those taking specific medications were excluded. Blood draws were postponed if the participant had used recreational drugs or received a vaccination within the last two weeks.
For the PBMC isolation, heparinized peripheral blood was diluted with 1X PBS in a 1:1 ratio. In 50-ml conical tubes, 30 ml of the diluted blood was carefully layered over 15 ml of lymphocyte separation solution (Wisent Bioproducts, CA). The samples were centrifuged at 624g for 30 minutes at room temperature. Mononuclear cells were then collected into another 50-ml tube and centrifuged at 433g for 15 minutes with 25 ml of PBS. After discarding the supernatant, the pellet was washed with 25 ml of PBS and centrifuged again at 400g for 12 minutes. The PBMCs were cultured in a medium comprising 10% heat-inactivated fetal bovine serum (FBS; Wisent Inc., Montreal, QC, Canada) in Roswell Park Memorial Institute (RPMI 1640) medium, supplemented with 1mM penicillin, streptomycin, and 2mM GlutaPlus (Wisent Inc., QC, Canada) at 37° cell culturing.