Transfectin

The cell lines Huh-7 and Huh-7 NS3-3′ were transfected as previously reported [14 (link),15 (link)]. To assess the inhibitory activity of the chimeric RNA molecules ex vivo on HCV IRES-dependent translation, Huh-7 cells were used as model system. 90,000 cells/well were seeded onto a 24-well plate and allowed to reach 80–90% confluence (36–48 h). A mix containing 1 µg of the RNA construct IRES-FLuc and 100 ng of cap-RLuc was supplemented with 5 µg of each chimeric inhibitor or the non-related RNA80. TransFectin™ (Bio-Rad, Hercules, CA, USA) was used as the transfection reagent following the manufacturer’s instructions. Luciferase activity was detected 18 h after transfection using the Dual-Luciferase™ reporter assay system (Promega).
Analysis of the inhibitory effect on HCV replication was performed by transfecting Huh-7-NS3-3′ cells with the different chimeric inhibitors under study. At 24 h before transfection, 90,000 cells were seeded and grown until 80% confluence in 1.5 cm diameter dishes in the presence of culture medium without G-418. The cells were then transfected with 4 µg of each inhibitor RNA or RNA80 using TransFectin™ (Bio-Rad). At 24 h post-transfection, the cells were harvested and processed for further RNA extraction.

Neuronal transfections were carried out at 13–14 DIV using transfectin (BioRad, Hercules, CA, USA), according to the manufacturers’ instructions (DNA: transfectin ratio 1 µg:3 µL), with 1–2 µg of plasmid DNA per 20 mm well. Simple transfections of NKCC1-HA-Flag-mVenus or KCC2-Flag were carried out with a plasmid concentration of 1 µg. The following ratios of plasmid DNA were used in co-transfection experiments: 0.4:0.4:0.4 µg for NKCC1a/b and KCC2 constructs together with GPHN.FingR-eGFP or homer1c-GFP for the quantification of clustering of NKCC1a/1b and NKCC1a/1b-KCC2 at synapses; 1:0.4:0.4 µg for NKCC1a/b constructs together with GPHN.FingR-eGFP and homer1c-DsRed for single-particle tracking (SPT) at synapses; 1:0.2 µg for NKCC1a/b or KCC2 constructs with eGFP for SPT on the AIS, axon and dendrite; 0.7:0.7 µg for NKCC1a/b or KCC2 constructs with clathrin-YFP or GPHN.FingR-eGFP for SPT in endocytic zones and STORM experiments, respectively; and 0.7:0.7 µg for NKCC1a/b or KCC2 constructs with dendra2-gephyrin for STORM/PALM. Experiments were performed 7–10 days post-transfection. SPT, STORM and STORM/PALM experiments were performed with Δflag-ΔmVenus NKCC1 constructs. Standard epifluorescence microscopy was performed with Flag-mVenus NKCC1 constructs.

For SILAC proteomic analyses, 8505C cells growth in “light” SILAC media were transfected with siRNA specific for Bag3 (siBAG3#1: 5′-AAGGUUCAGACCAUCUUGGAA-3′) or scrambled siRNA (siSCR#1: 5′-CAGUCGCGUUUG CGACUGG-3′) (synthesized by GE Healthcare Dharmacon, Buckinghamshire, UK) and used at a final concentration of 200 nM. 8505C cells growth in “light” media were plated at 30-40% confluence and transfected with siRNAs using Transfectin (Bio-Rad, Hercules, CA, USA) according to manufacturer's instructions. 8505C cells growth in “heavy” SILAC medium were used as non-transfected control. Transfections were carried out in triplicate. Cells were harvested 72 h after transfection and equal amount of “light” (siBAG3 or siScrambled transfected cells) and “heavy” cells were pooled together.
Other small-interfering RNA used were: siBAG3#2 (Cat. No. SR306333), siSERPINB2 (Cat. No. SR303346), siCAV1 (Cat. No. SR300603) (synthesized by OriGene Technologies, Rockville, MD, USA), used according to the manufacturer's recommendations. The scrambled nonsense siRNA (siSCR#2) (Universal scrambled negative control siRNA duplex, Cat. No. SR30004), which has no homology to any known gene, was used as control. Cell transfection with siRNA oligonucleotides was performed using Transfectin (Bio-Rad) according to manufacturer's recommendations and as above described.