On targetplus smart pool gem sirna l 008717 00 0020

One million HTLV-1-infected cells (C91/PL) were transfected with 75 nM of Gem siRNA (ON-TARGETplus smart Pool Gem siRNA L-008717-00-0020, Thermo Scientific) or control siRNA (ON-TARGETplus Nontargeting Pool, Thermo Scientific) using Hiperfect reagent (Qiagen). Twenty-four, forty-eight or seventy-two hours post-transfection, supernatants were collected and centrifuged at 2 000 rpm for 3 min. Supernatants were diluted in RPMI complete media (1∶100) and p19 levels were assessed using the RETROtek HTLV p19 Antigen ELISA kit (Zeptometrix Corporation) following manufacturer instructions.

MOLT4 or Jurkat cell lines were transduced with control (Lenti-IRES-GFP) or HA-Gem lentiviral particles (MOI = 5). Forty-eight hours later, 5×10⁵ transduced cells were loaded in the upper chamber of 5 µm polycarbonate transwell filters (Corning). Complete RPMI medium containing or not SDF1/CXCL12 chemo-attractant (150 ng/ml) was added in the lower chamber. Twenty-four hours later, quantification of the number of migrating cells was performed using TruCount flow tubes (BD Biosciences) and flow cytometer Facscalibur4c+HTS (BD biosciences).
HTLV-1-infected cells (C91/PL, Hut102) or non-infected control cells (Jurkat, MOLT4) were transfected with 75 nM of Gem siRNA (ON-TARGETplus smart Pool Gem siRNA L-008717-00-0020, Thermo Scientific) or siRNA control (ON-TARGETplus Non-targeting Pool, Thermo Scientific) using Hiperfect reagent (Qiagen). 5×10⁵ transfected cells were loaded in the upper chamber of 5 µm polycarbonate transwell filters (Corning) whereas complete RPMI medium was added in the lower chamber. Quantification was performed as described above.