The shRNA lentiviral vectors used for knockdown of DDX27 were synthesized by Genepharma Company (Suzhou, China). The following shRNA sequences were used for the DDX27 knockdown experiment: shRNA1 (5′-GCTTGCGGACCTCGGCTTAAT-3′), shRNA2 (5′-GCTGATTTCAACCCTGATTTC-3′), shRNA3 (5-GGAGAAAGAAGCAAAGGAAGG-3′), and the negative control (5′-TTCTCCGAACGTGTCACGT-3′). We transfected cell lines with lentiviruses carrying these shRNA plasmids according to the manufacturer’s instructions. shRNA3 was chosen as the representative shRNA because it was the most efficient one. The evidence in the protein level change of knockdown of DDX27 by shRNA3 sequences in HCT116 and HT29 lines is provided in Figure S1 .
Shrna lentiviral vectors
Manufactured by GenePharma
Sourced in China
ShRNA lentiviral vectors are a type of gene delivery system used for the stable expression of short hairpin RNA (shRNA) in target cells. They are designed to efficiently transduce a wide range of cell types and enable long-term, inducible knockdown of gene expression.
Automatically generated - may contain errors
Lab products found in correlation
Cytodex 3, by GE Healthcare (1 mentions)
Hrp conjugated secondary antibody, by Jackson ImmunoResearch (1 mentions)
4 6 diamidino 2 phenylindole (dapi), by Santa Cruz Biotechnology (1 mentions)
Lipofectamine 3000, by Thermo Fisher Scientific (1 mentions)
Pspax2, by Addgene (1 mentions)
Pmd2 g, by Addgene (1 mentions)
Polybrene, by GenePharma (1 mentions)
Lentiviral vector, by GenePharma (1 mentions)
4 protocols using shrna lentiviral vectors
1
Lentiviral shRNA Knockdown of DDX27
2
Rho GTPase Knockdown Pathway Evaluation
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The shRNA lentiviral vectors that were used to knockdown of RhoC expression were purchased from GenePharma (Suzhou, China). An anti‐RhoC antibody was purchased from Abcam (Cambridge, MA, USA). The anti‐mitogen‐activated protein kinase (MAPK) and anti‐Rho‐associated coiled‐coil kinase (ROCK) antibodies were purchased from Proteintech (Chicago, IL, USA). Phalloidin was purchased from Cytoskeleton (Denver, CO, USA). DAPI was purchased from Santa Cruz Biotechnology (Dallas, Texas, USA). Ezol was purchased from GenePharma; Cytodex3 was purchased from GE Healthcare (Uppsala, Sweden), and HRP‐conjugated secondary antibody was purchased from Jackson (West Grove, PA, USA).
3
Lentiviral Transduction of Glioma Stem Cells
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Lentiviral shRNA vectors were obtained from GenePharma (Jiangsu, China). For OV experiments, lentiviral vectors expressing circEZH2, circEZH2 del-ATG and EZH2-92aa-3×Flag ORF were cotransfected with the packaging plasmid psPAX2 (Addgene) and the envelope plasmid pMD2.G (Addgene) into 293T cells using Lipofectamine 3000 (Invitrogen) for lentivirus production. To establish stable cell lines, GSCs were transduced with the above lentiviral vectors in culture medium containing 8 μg/ml polybrene (GenePharma). After 24 h of incubation, cells were screened with 2 μg/ml puromycin for 3 days.
4
Myoferlin Knockdown and Overexpression
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MYOF was knocked down in NCI-N87 and MKN45 cells using lentiviral shRNA vectors (GenePharma Technology) designed to target the sequences 5′-GAAAGAGCTGTGCATTATAAA-3’ and 5′-GCTGTGGAGAAGAAGTTTAAC-3′ in the MYOF coding region. Stable knockdown cells were isolated by selecting for puromycin resistance.
To overexpress MYOF, human MYOF complementary DNA (cDNA) was amplified and inserted into a lentiviral vector; as a control, an empty lentiviral vector (GenePharma Technology) was prepared similarly. The vectors were introduced into HGC27 and SNU1 cells, and cells were selected for puromycin resistance 2 weeks before experiments performed as previously described (25 (link)).
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MYOF was knocked down in NCI-N87 and MKN45 cells using lentiviral shRNA vectors (GenePharma Technology) designed to target the sequences 5′-GAAAGAGCTGTGCATTATAAA-3’ and 5′-GCTGTGGAGAAGAAGTTTAAC-3′ in the MYOF coding region. Stable knockdown cells were isolated by selecting for puromycin resistance.
To overexpress MYOF, human MYOF complementary DNA (cDNA) was amplified and inserted into a lentiviral vector; as a control, an empty lentiviral vector (GenePharma Technology) was prepared similarly. The vectors were introduced into HGC27 and SNU1 cells, and cells were selected for puromycin resistance 2 weeks before experiments performed as previously described (25 (link)).
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