Double-strand oligonucleotides corresponding to the target sequences were cloned into the pSuper-Retro plasmid (Oligoengine). The following sequences were targeted for human GPATCH3 mRNA: 1#: 5’-GCAAGCGTGGATTGGGGTA-3’; 2#: 5’-CCTACCTGGCAGATATACC-3’; 3#: 5’-GTGAAGAAATACCCCAAGG-3’. Small interfering RNAs (siRNAs) targeting human GPATCH3 were purchased from Ribobio. 1# siRNA: 5’-GGAACAGAGACTCCGAGAT-3’, 2# siRNA: 5’-GTACCATGGAGAGAAGCTA-3’. siRNA were delivered into A549s and HFFs by PepMute siRNA transfection reagent (SignaGen) according to procedures recommended by the manufacturer.
Psuper retro plasmid
Manufactured by Oligoengine
Sourced in United States
PSUPER.retro plasmid is a lab equipment product designed for molecular biology applications. It functions as a vector for cloning and expressing short hairpin RNA (shRNA) sequences in mammalian cells. The product enables the construction of retroviral shRNA expression systems.
Automatically generated - may contain errors
Lab products found in correlation
Opti mem medium, by Thermo Fisher Scientific (2 mentions)
Pepmute sirna transfection reagent, by SignaGen (1 mentions)
Sirnas, by RiboBio (1 mentions)
Lipofectamine 2000 reagent, by Thermo Fisher Scientific (1 mentions)
Mt yfp, by Takara Bio (1 mentions)
Dulbecco modified eagle medium (dmem), by Thermo Fisher Scientific (1 mentions)
Lipofectamine 2000 transfection reagent, by Thermo Fisher Scientific (1 mentions)
4 protocols using psuper retro plasmid
1
Silencing GPATCH3 in Human Cell Lines
2
ACSL4 knockdown in MDA-MB-231 cells
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The MDA-MB-231 cell line was used for stable transfection with pSUPER.retro plasmid (OligoEngine, Seattle, WA, USA) containing shRNA ACSL4 (5′AAGATTATTCTGTGGATGA-3′) or the empty vectors as control in Opti-MEM medium and Lipofectamine 2000 reagent (Invitrogen) as previously described by for our group [5 (link)]. ACSL4 knockdown was evaluated through Western blot (Supplementary Fig. 1B ).
3
Mitofusin 2 Silencing Protocol
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The specific primers used for real-time PCR were: human Mitofusin 2 cDNA forward, 5′-ATGCATCCCCACTTAAGC -3′ and reverse, 5′-GTTCTTCTGTGGTAACGG -3′; and human We used a murine Mfn2-shRNA which has been already proved in our previous works and reported [10] (link). BLAST data showed that the 19-mer RNAi sequence is specific against the mRNA sequence of human Mfn2 with 100% identity with the Mfn2 transcript of Homo sapiens, without targeting Mfn1, the other isoform that participates in mitochondrial fusion. We also designed a human Mfn2-specific shRNA. We used pSUPER.retro plasmid (OligoEngine, Seattle, WA, USA) containing a 19-bp DNA fragment of the human Mfn2 (GGAAGACATTGAGTTCCAT) named hMfn2-shRNA in the adequate frame shift to generate a shRNA.
4
Evaluating Mitochondrial Dynamics in WJ-MSCs
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In order to evaluate mitochondrial presence and transport, WJ-MSCs (5 × 10 5 cells/well) were grown up to 50%-70% confluence onto cover glass dishes and mitochondria were labeled with a mitochondrial target sequence linked to a fluorescent dye. Transfection was performed with a reagent mixture including 0.8 μg pSUPER-retro plasmid (Oligoengine,VEC-PRT-0001) containing the mitochondrial target sequence constituted by a cytochrome oxidase subunit and 0.8 μg yellow fluorescence protein mt-YFP (Clontech, 632,443) in Opti-MEM medium (Life Technologies, 31,985-070), adding 2 μl lipofectamine 2000 transfection reagent (Invitrogen, 11,668-019) according to the manufacturer's instructions. Cells were incubated with this mix in DMEM (Life Technologies, 12,800-082) supplemented with 10% v/v fetal bovine serum in PBS without antibiotics for 5 h at 37 °C and 5% CO 2 . Culture medium was then replaced by one supplemented with 10% v/v fetal bovine serum in PBS and antibiotics (100 U/ml penicillin/streptomycin, 0.3 μg/ml gentamicin and 50 μg/ml fungizone) under the same conditions for 48 h.
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