PMC:4385186 / 11374-13991
Annnotations
{"target":"https://pubannotation.org/docs/sourcedb/PMC/sourceid/4385186","sourcedb":"PMC","sourceid":"4385186","source_url":"https://www.ncbi.nlm.nih.gov/pmc/4385186","text":"Knockdown and Overexpression of Genes of Interest in ESCC Lines\nLentiviral vector pLKO.1-puro and its packaging plasmids pMD2.G and psPAX2 were obtained from Addgene. Knockdown experiments of the special genes were performed in at least two ESCC lines with high endogenous protein levels. Two independent short hairpin RNAs (shRNAs) were cloned into the pLKO.1-puro vector as described previously.13 Specifically, to produce virus production, titration, and infection lentiviruses, we transfected HEK293T cells with the packaging plasmids along with the lentiviral shRNA vector by using Lipofectamine 2000 reagent (Invitrogen) according to the manufacturer’s instructions, and we changed the medium after 6 hr. Virus was harvested 24 hr after transfection, passed through 0.22-μm filters, and used fresh for shRNA infection. To perform lentiviral infections, we plated the target ESCC cells at 40%–50% confluence and incubated them overnight (16 hr). On the day of infections, the culture medium was replaced with the appropriately titered viral supernatant (1.5 ml/well) and incubated at 37°C for 24 hr; afterward, the viral supernatant was replaced with fresh media. Forty-eight hours later, infected cell populations were selected in puromycin (2 μg/ml). After 5 days of selection, shRNA-knockdown efficiency was determined by immunoblot analysis for the respective proteins with the use of specific antibodies. For knockdown of special genes in ESCC cells, two independent shRNA constructs that had been cloned into the pLKO.1-puro vector were used (Table S5). A non-specific targeting shRNA was also cloned into the pLKO.1-puro vector with the use of a scrambled control (SCR). Relative amounts of special gene product were normalized to β-actin levels.\npMSCV-puro empty vector and wild-type pMSCV-puro-ZNF750 were generous gifts from Paul A. Khavari (Stanford University). pcDNA3-RFP empty vector and wild-type pcDNA3-RFP-AJUBA (MIM 609066) were generous gifts from Alejandra Garcia-Cattaneo (National Heart and Lung Institute, Imperial College London). The wild-type versions of these genes of interest were cloned into the pLV-EGFP(2A)-puro-GFP vector and validated by sequencing. For overexpression experiments, we used the pLV-EGFP(2A)-puro-GFP vector as a control. Viruses were produced as previously described. ESCC cells with low endogenous protein levels were infected with viruses as previously described.14 Twenty-four hours after infection, cells were subjected to subsequent experiments. The mutants of genes of interest were generated with the QuikChange II Site-Directed Mutagenesis Kit (Agilent).","divisions":[{"label":"title","span":{"begin":0,"end":63}},{"label":"p","span":{"begin":64,"end":1758}}],"tracks":[{"project":"2_test","denotations":[{"id":"25839328-19922768-2052569","span":{"begin":397,"end":399},"obj":"19922768"},{"id":"25839328-22430208-2052570","span":{"begin":2420,"end":2422},"obj":"22430208"}],"attributes":[{"subj":"25839328-19922768-2052569","pred":"source","obj":"2_test"},{"subj":"25839328-22430208-2052570","pred":"source","obj":"2_test"}]}],"config":{"attribute types":[{"pred":"source","value type":"selection","values":[{"id":"2_test","color":"#c893ec","default":true}]}]}}