Psuper retro vector

The target sequence [sense 5′-AAAGACAGCCTTAGCTTTGAG-3′] for human CIB1 RNAi was selected with the use of the siRNA target finder program of Ambion. The annealed oligonucleotides including sense and anti-sense strands of the target sequence were inserted into the pSUPER.retro vector (OligoEngine). As a control, annealed oligonucleotides containing a target sequence (sense 5′-GGCTACGTCCAGGAGCGCACC-3′) for a GFP shRNA were inserted into the same vector. The nucleotide sequences of the various inserts were confirmed by DNA sequencing. SH-SY5Y cells were transfected with pSUPER.retro vectors encoding either the control (GFP) or CIB1 shRNA, and stable transfectants were selected in the presence of puromycin (0.25 μg/ml). Rat CIB1 siRNA (sense 5′-AAGAGTCACTGCATACCCGAG-3′), Mouse ASK1 siRNA (sense 5′-AATTGCAGTCTGCACAGCCTTTCGG-3′), or control GFP siRNA (sense 5′-GGCTACGTCCAGGAGCGCACC-3′) oligonucleotides were obtained from Invitrogen and introduced into primary rat mesencephalic dopaminergic neurons by transfection for 48 h with the use of RNAiFect (Qiagen).

Oligonucleotides containing siRNA-expressing sequence targeting SMARCD1 were annealed (shSMARCD1 top: 5’-GATCCCCGCAGATCTTTGAGTCTCAACGTTCGAAGAGCGTTGAGACTCAAAGATCTGCTTTTTA-3’, shSMRCD1 bottom: 5’-AGCTTAAAAAGCAGATCTTTGAGTCTCAACGCTCTTCGAACGTTGAGACTCAAAGATCTGCGGG-3’; shSMARCD2 top: 5’-GATCCCCGCAGGGACCTCAAGACAATGATTCGAAGAGTCATTGTCTTGAGGTCCCTGCTTTTTA-3’, shSMARCD1 bottom: 5’-AGCTTAAAAAGCAGGGACCTCAAGACAATGACTCTTCGAATCATTGTCTTGAGGTCCCTGCGGG-3’), and cloned into the pSUPER.retro vector (OligoEngine, Seatle, WA, USA).

The HA-tagged MT3 plasmid or Myc-tagged PPARγ plasmid were constructed in a CMV promoter-derived mammalian expression vector (pCS4+). For MT3 knockdown experiments, oligonucleotides for small hairpin RNA (shRNA) were generated by targeting a 19-base pair sequence of the mouse MT3 gene. shMT3#1: sense 5′-GAT CCC CCC AAG GAC TGT GTG TGC AAT TCA AGA GAT TGC ACA CACA GTC CTT GGT TTTTA-3′, antisense 5′-AGC TTA AAA ACC AAG GAC TGT GTG TGC AAT CTC TTG AAT TGC ACA CAC AGT CCT TGG GGG-3′; shMT3#2: sense 5′-GAT CCC CGC AAG TGC AAG GGC TGC AAA TTT CAA GAG AAT TTG CAG CCC TTG CAC TTG C TTTTTA-3′, antisense 5′-AGC TTA AAA AGC AAG TGC AAG GGC TGC AAA TTC TCT TGA AAT TTG CAG CCC TTG CAC TTG CGGG-3′. Sense and antisense oligonucleotides were annealed and ligated into a pSuper-retro vector (Oligoengine, Seattle, WA, USA). Both overexpression and knockdown experiments in the HEK 293T cells and 3T3-L1 cells were performed by using polyethyleneimine (PEI) (Polysciences, Inc., Warrington, PA, USA).

For PLD1 targeting, previously described oligos were used,^{50 (link)} and knockdown efficiency was assessed.^{51 (link)} For DGKζ targeting, we used the previously validated sequences 5′-CUAUGUGACUGAAGAUCGCATT-3′ and 5′-GGUGAAGA GCUGAUUGAGGTT-3′.^{22 (link), 38 (link), 52 (link)} DGKα was silenced with validated sequences;^{32 (link)} either a scrambled (Ambion, Austin, TX, USA) or the equivalent mouse sequence was used as control. For transient targeting, distinct siRNAs were transfected in cells using Oligofectamine (Invitrogen, Carlsbad, CA, USA). For stable targeting, sequences that target murine (control) or human DGKζ were cloned in the pSuperRetro vector (Oligoengine, Seattle, WA, USA), and SW480 cells stably expressing shRNAi were obtained by infection with pSuperRetro-cloned sequence-containing retroviruses using standard protocols.

Oligonucleotides containing the siRNA-expressing sequence targeting ATP6V0A2 were annealed (shATPV0A2 top: 5′-GATCCCCGCAGCTTTGACGTGACCAACATTCGAAGAGTGTTGGTCACGTCAAAGCTGCTTTTTA-3′, shATP6V0A2 bottom: 5′-AGCTTAAAAAGCAGCTTTGACGTGACCAACACTCTTCGAATGTTGGTCACGTCAAAGCTGCGGG-3′), and cloned into the pSUPER.retro vector (OligoEngine, Seattle, WA). Preparation of viral supernatant and infection of target cells was performed as described26 (link). Transduced cells were selected with 2 μg/mL puromycin for 3 days.

DGK-targeting was performed using the human DGKα (nt 1153-1173) and DGKζ (nt 2290-2310) validated sequences [35 (link), 36 (link), 71 (link)]. For transient silencing the sequences were transfected as dsRNA (Ambion) using oligofectamine (Invitrogen). A scramble dsRNA was used as control (Ambion). Effective DGK depletion was achieved at 72-96 h post-transfection. After 96h the expression was recovered. For stable silencing, 64 pb double strand oligonucleotides, encompassing the corresponding interfering 21 nt sequence and a hairpin structure, were cloned in the pSuperRetro vector (Oligoengine). As control the pSuperRetro construct harboring the homolog specific DGKζ mouse sequence was used. This control did not reduce the levels of human DGKα or ζ. SW480 cells were infected using standard protocols with the pSuperRetro-cloned sequence-containing retroviruses. Cells with the retrovirus insertion were selected by culturing in media with G418 (800 μg/ml) until the control, non-infected cells died (~1 week). The pools were checked for DGK silencing, maintained in media with G418 (500 μg/ml) and used in the long-term experiments.

Mammalian expression vectors for human Flag- or HA-tagged RIG-I, RIG-I-N (containing only the N-terminal CARD domain, 1 to 284 amino acids), MAVS, TBK1, IKKε, IRF3 and its mutant IRF3-5D (the phosphorylation sites as Ser396, Ser398, Ser402, Thr404, and Ser405 residues were replaced by aspartic acid in the C-terminal end of IRF-3) (47 (link)), ubiquitin and its mutants K48 and K63 ubiquitin, IFN-β promoter luciferase reporter, ISRE, and NF-κB luciferase reporter constructs were described previously (10 (link), 39 (link), 48 (link)). The full-length human MAP4K1 was cloned into mammalian expression vector pRK5 with an N-terminal HA or Flag tag and pcDNA3.1 with an N-terminal Myc tag by standard molecular biology techniques. MAP4K1-specific siRNA constructs were generated by the pSuper.retro vector (OligoEngine, Seattle, WA, USA) with double-strand oligonucleotides targeting MAP4K1 sequences using protocols recommended by the manufacturer. The following sequences were designed for targeting human MAP4K1 mRNA: MAP4K1-RNAi 1, 5′-CCTGGATCTTCTTGACAAA-3′; MAP4K1 RNAi 2, 5′-CCTGATCCTGGATCTTCTT-3′, and MAP4K1 RNAi 3, 5′-CCAGACGCCTCTCTTTCAT-3′. The following shRNA sequences were used: control shRNA sequence (5′-CAACAAGATGAAGAGCACCAA-3′) and Dtx4 shRNA (5′-TTAAGGCAGCCGTGGTCAATG-3′).