For overexpression of GDF15, the GDF15 transcript was cloned into a pLenti6/V5-TOPO vector (Life Technologies Corporation, Carlsbad, CA). The p16 and p53 shRNA retroviruses were prepared by transfection of pRetroSuper-p53sh and pRetroSuper-p16sh vectors into Human Embryonic Kidney 293 (HEK 293T) cells. After incubation for 3 days, the media were collected and centrifuged at 1,650 × g for 10 min. The viral solution was filtered using 0.45-μm filter membranes and concentrated with Vivaspin® 20 centrifugal concentrators (Sartorius, Göttingen, Germany). Cells were then transduced with either the p16 shRNA or the p53 shRNA retrovirus. After incubation for 48 h, the p16 or p53 shRNA retrovirus-transduced cells were treated with 100 mg/ml recombinant GDF15 protein for 4 days.
Plenti6 v5 topo vector
Manufactured by Thermo Fisher Scientific
Sourced in United States
The PLenti6/V5-TOPO vector is a lentiviral expression vector designed for the efficient cloning and expression of genes in mammalian cells. It provides a simple and convenient method for the directional cloning of blunt-end PCR products. The vector includes a V5 epitope tag for the detection and purification of the expressed protein, as well as a blasticidin resistance gene for the selection of transduced cells.
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7 protocols using plenti6 v5 topo vector
1
Overexpression of GDF15 in Cells
2
Lentiviral Overexpression and Knockdown
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For XAF1 overexpression, the XAF1 transcript was cloned into a pLenti6/V5-TOPO vector (Life Technologies Corporation, Carlsbad, CA, USA). Cells were transduced with XAF1 lentivirus for 48 h. p16 and p53 shRNA retroviruses were prepared by transfection of the pRetroSuper-p53sh and pRetroSuper-p16sh vectors. After incubation for 3 days, media were collected and centrifuged at 1,650 g for 10 min. The viral solution was filtered with 0.45-μm filter membranes and concentrated with a Vivaspin 20 (Sartorius, Goettingen, Germany).
3
Plasmid Construction and Cell Transduction
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The shATG5 (hU6-MCS-Ubiquitin-EGFP-IRES-puromycin, GOSL0195542) Plasmid was constructed by GENECHEM, and cells were selected in puromycin. GFP-tagged CHD1L was cloned into pLenti6/V5-TOPO®vector (Life technologies, Carlsbad, CA), and cells were selected with Blasticidin (Sigma-Aldrich, St. Louis, MO). ZKSCAN3 expression construct (ZKSCAN3 [NM_001242894.1]/FLAG was cloned into pLV[Exp] -mCherry:T2A:Puro-EF1A) was constructed by Cyagen Biosciences Inc, and cells were selected with Ampicillin. For transient knockdown assay (siRNAs are listed in Supplementary Table 2 ), the siRNA against ZKSCAN3, Paxillin, and the scrambled sequence (Qiagen, CA, USA) was transfected to the indicated cells. Virus production and cell transduction in indicated cells were performed as previously described [6 (link)].
4
Modulating MYLK Expression in Cells
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Cells were cultivated to 50–60% confluence and were then treated with siRNA or shRNA (sequences of used siRNA and shRNA was listed in Table S1 ) according to the manufacturer’s instructions. For overexpression of nmMYLK (MYLK-GFP plasmid presented by Addgene), cells were harvested in 48 h after transfection. For MYLK knockdown assay, green fluorescent protein (GFP)-tagged shMYLK was cloned into pLenti6/V5-TOPO vector (Life Technologies) and the viruses were packaged and transfected into HepG2 cells according to the manufacturer’s instructions.
5
Generation of Caspase-11 Expressing Cell Lines
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Bone marrow-derived macrophages (BMDMs) were cultured as previously described77 (link). Construction of red fluorescent caspase-11 and mutant caspase-11 using the pLenti6/V5 TOPO vector (Invitrogen Life Technologies) was previously described78 (link). To generate HEK293 cells stably expressing red fluorescent caspase-11 and mutant caspase-11. Cells were transduced with lentivirus at an MOI of 1 in the presence of 6 μg/mL polybrene for 8–16 hrs. Afterwards, cell culture media was replaced and cells were selected with blasticidin (5 μg/mL) for 10 days resulting in a homogeneous yield of stably transduced cell line.
6
Lentiviral Vectors for MyD88 and Snail Modulation
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Lentiviral vectors encoding the human MyD88 gene were generated by using pLenti6/V5-TOPO vector (Invitrogen, Carlsbad, CA, USA) and were designated as LV-MyD88. The empty vector was used as a negative control and was designated as LV-GFP. Lentiviral vectors encoding short hairpin RNAs targeting MyD88 or scramble shRNA were generated by using pLenti6/BLOCKiT-DEST (Invitrogen) as previously described,13 (link) and were designated as shMyD88 and shNon, respectively. Two target sites were designed for knockdown of MyD88 expression. Further details are available in the Supplementary Data .
siRNA targeting specific human Snail sites (siSnail-1 and siSnail-2) and negative control siRNA were generated by GenePharma (Shanghai, PR China). Details are available in theSupplementary Data (Supplementary Table S2 ).
Δp85 (Addgene, Cambridge, MA, USA; plasmid 13432), with adominant negative p85 alpha and deleted p110 alpha binding domain, disenables PI3-K to phosphorylate Akt. DN-Akt (Addgene, plasmid 9031) was built by generating a vector backbone (pcDNA3.0) with a mutation that deprived Akt of its phosphorylation ability. Ser9A-GSK-3β (Addgene, plasmid 14754), was similarly established by generating pcDNA3.0 with a mutation that was a constitutively active mutant blocking phosphorylation of GSK-3β.
siRNA targeting specific human Snail sites (siSnail-1 and siSnail-2) and negative control siRNA were generated by GenePharma (Shanghai, PR China). Details are available in the
Δp85 (Addgene, Cambridge, MA, USA; plasmid 13432), with adominant negative p85 alpha and deleted p110 alpha binding domain, disenables PI3-K to phosphorylate Akt. DN-Akt (Addgene, plasmid 9031) was built by generating a vector backbone (pcDNA3.0) with a mutation that deprived Akt of its phosphorylation ability. Ser9A-GSK-3β (Addgene, plasmid 14754), was similarly established by generating pcDNA3.0 with a mutation that was a constitutively active mutant blocking phosphorylation of GSK-3β.
7
Lentivirus-mediated AKR7A3 Overexpression and Knockdown
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The coding region sequence of AKR7A3 was amplified by PCR and cloned into pLenti6/V5-TOPO vector (Invitrogen). Empty vector or AKR7A3 constructs were co-transfected with the ViraPower Lentiviral packaging plasmids (Invitrogen) into 293FT cells. Supernatant containing lentivirus was added to QGY7703 and PLC8024 cells. Blasticidin (Invitrogen) was added to the cells for screening for stably transduced cells after 24 hours.
For knockdown of AKR7A3, plasmids of two short hairpin RNAs targeting AKR7A3 and scrambled control were purchased from GeneCopoeia (Rockville). These plasmids were packed into virus and transduced into MHCC-97L and H2M cell lines. Puromycin was used to screen for stably transfected cells.
For knockdown of AKR7A3, plasmids of two short hairpin RNAs targeting AKR7A3 and scrambled control were purchased from GeneCopoeia (Rockville). These plasmids were packed into virus and transduced into MHCC-97L and H2M cell lines. Puromycin was used to screen for stably transfected cells.
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