Stable knockdown of TUSC4 expression was established via RNA interference using lentiviral vector short hairpin RNA (Sigma; MISSION; NM660545). TUSC4 was targeted with a lentiviral particle of MISSION short hairpin RNA as well as MISSION nontargeted control particles. Western blotting was performed after transduction to confirm the knockdown efficiency, and puromycin was added to U2OS cell medium to maintain the TUSC4-knockdown specificity. For transient transfection, human TUSC4 siRNA was purchased from Thermo Scientific (On-Target; 10641), and the TUSC4 target sequences were GCAUCGAACACAAGAAGUA and GACCCAAGAUCACCUAUCA. Human BRCA1 siRNA was purchased from Thermo Scientific (On-Target; J-003461-09), and the BRCA1 target sequence was CAACAUGCCCACAGAUCAA. Human Herc2 siRNA was purchased from Thermo Scientific (On-Target; J-007180-09, J-007180-10, J-007180-11, and J-007180-12), and the Herc2 target sequences were 5′-GCACAGUAUCACAGGUA-3′, 5′-CGAUGAAGGUUUGGUAUUU-3′, 5′-GAUAAUACGACACAGCUAA-3′, and 5′-GCAGAUGUGUGCUAAGAUG-3′, respectively.
On target
Manufactured by Thermo Fisher Scientific
Sourced in Germany
On-Target is a versatile laboratory instrument designed for a wide range of applications. It provides precise and reliable performance for tasks such as sample preparation, analysis, and data processing. The core function of On-Target is to enable accurate and efficient laboratory workflows.
Automatically generated - may contain errors
Lab products found in correlation
Dharmafect 4, by Thermo Fisher Scientific (2 mentions)
Imdm culture medium, by Thermo Fisher Scientific (2 mentions)
Amaxa nucleofector, by Lonza (1 mentions)
Smartpool, by Thermo Fisher Scientific (1 mentions)
Lipofectamine rnaimax reagent, by Thermo Fisher Scientific (1 mentions)
Smartpool on target, by Thermo Fisher Scientific (1 mentions)
Hiperfect transfection reagent, by Qiagen (1 mentions)
Siarnt, by Thermo Fisher Scientific (1 mentions)
8 protocols using on target
1
Lentiviral Knockdown and Transient Silencing of TUSC4, BRCA1, and HERC2
2
siRNA and shRNA Experiments Protocol
3
Knockdown of CYP3A5, HNF4A, and NR1I2 in PACO cells
4
siRNA Transfection of Myeloma Cells
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Cells (1 × 106/transfection) were transfected with siRNA using an Amaxa Nucleofector (Lonza, Basel, Switzerland) according to the manufacturer’s protocols as described previously20 (link). MMCs were trypsinized and resuspended in Basic Nucleofection Solution at 1 × 107/ml. Subsequently, 100 μl of cell suspension (1 × 106 cells) was mixed with 15 nM of ON-TARGET plus siRNA or negative controls (Thermo Fischer Scientific Inc., Waltham, MA) as indicated. Transfected cells were harvested for RNA and protein isolation at indicated times.
5
siRNA and shRNA Experiments Protocol
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For siRNA experiments, ON-TARGET plus siRNAs SMARTpools (Thermo) were used. For small hairpin RNA (shRNA) experiments, all the pGIPZ lentivirus shRNA plasmids were purchased from the C-BASS Core at Baylor College of Medicine. Details are in the Supplemental Information .
6
Knockdown of CYP3A5, HNF4A, and NR1I2 in PACO cells
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PACO cells were grown to 80% confluency. The transfection reagent Dharmafect 4 (Thermo Scientific), nOn-Targeting (NT-control) and CYP3A5, HNF4A or NR1I2 siRNA (On-Target plus SMARTpool Thermo Scientific, Supplementary Table 12 ) were pre-incubated at RT for 5 min at a ratio of 1:4 in IMDM culture medium (Gibco). For the HNF4A and NR1I2 double knockdown, the individual siRNAs were pre-incubated together at a ratio of 1:8 in IMDM culture medium (Gibco). Dharmafect 4 was then combined with the siRNAs and incubated for further 20 min at RT. The mixture was then added to the PACO culture medium. The culture medium was aspirated and the transfection agent-RNA complex mixture was added to the monolayer. Flasks were incubated at 37 °C for 72 h until further analysis.
7
Regulation of SREBP-1c and ATF6α in HepG2 cells
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HepG2 cells were transfected with RNAi duplexes via RNAi MAX Lipofectamine reagent (Invitrogen, NY, USA). SREBP-1c was silenced in cells with ON-TARGET plus siRNAs SMARTpool (L-006891-00-0005) and parallel cells with ON-TARGET plus NON-TARGETing Pool (D-001810-10-05) at a concentration of 10 nM (Thermo Scientific). ATF6α was silenced in cells with duplex siRNAs (HSS177036) at a concentration of 10 nM (Invitrogen). Parallel cell cultures were transfected with equal concentrations (10 nM) of Stealth negative universal control (Invitrogen). At 24 hours after transfection, the medium was replaced with complete growth medium containing OA/PA (1 mM) and BBR (5 μM).
8
Arnt silencing in bone marrow-derived macrophages
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For transfection, isolated BMDMs were differentiated with 20 ng/ml M-CSF for 1 week and transfected afterwards with 150 nM SMARTpool ON-TARGET plus siArnt or ON-TARGET plus NON-TARGETing pool siCtrl (both Thermo Scientific, Karlsruhe, Germany) using Hiperfect transfection reagent (Quiagen, Hilden, Germany). Therefore, BMDMs were incubated in medium without FCS containing siRNA and Hiperfect. After 6 h, medium containing M-CSF (final concentration of 20 ng/ml) was added and after 24 h cells were stimulated with MCA (5 μg/ml) or dimethylsulfoxide (DMSO) as a control for 8 h.
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