To prepare LDHA-knockdown PMEL (PMELLDHA-KD) CD8+ T cells, shLDHA lentivirus particles were produced in HEK293T cells that were transfected with plasmids of pVSV-G, Delta 8.9 and pLKO.1 puro_shLDHA using the calcium phosphate method as described above. PMEL CD8+ T cells were primed for ...
ADAM17 (MSH026499-3-LVRU6GP) or scrambled control (CSHCTR001-LVRU6GP) were transected into dEC via lentivirus. The ratio of the shRNA-expressing cell was evaluated by fluorescence. Batches with over 90% of fluorescent cells were used for downstream analysis. ...
The supernatants containing virus were harvested 48 and 72 h after transfection and concentrated using Lenti-XTM concentrator (TaKaRa Bio). The viral pellet was resuspended in serum-free DMEM medium and viral titers were measured with QuickTiter Lentivirus Quantitation Kit (Cell Biolabs). Viral ...
resulting in Mast4 degradation. We then show that Mast4 stabilized by Wnt-mediated GSK-3β inhibition promotes β-catenin nuclear localization, ultimately increasing Runx2 transcriptional activity and subsequent osteogenic differentiation of MSCs. The effects of Mast4 on chondro-osteogenesis of mesenchymal...
TRPV4WT and TRPV4R269C with a C-terminal EGFP tag in pcDNA3.1 were subcloned into the lentivirus transfer vector FUGW (gift from David Baltimore, Addgene plasmid #14883) (PMID 11786607). In brief, TRPV4- EGFP was PCR amplified (Forward primer: CTTGGGCTGCAGGTCGACTCTA GAGATGGCGGATTCCAG...
For concentration of lentivirus, Retro-X concentrator was used according to the manufacturer's instructions (Clonetech). At 48 h after transduction, puromycin (1 mg ml À 1) was added to the medium to select transduced cells. FACS analysis. Cell cycle profiling of propidium iodide stained ...
The lentivirus particles were harvested, concentrated with Lenti-X Concentrator (Clonetech, Mountain View, USA) and used to infect KMS11. Transduced KMS11 were selected with 1 mg ml À 1 puromycin. The shRNA was induced from a tetracycline-inducible promoter with 1 mg ml À 1 doxycycline ...
Lentivirus was produced by transfecting HEK 293ET cells with the lentiviral vector plus the packaging plasmids pCMVΔR8.91 and pMD.G in a ratio of 10:7:3, using TransIT-293 Transfection Reagent. Harvest of viral supernatants, lentiviral transductions and selection for stable expression (150 μ...
WIBR3 hESCs maintained in primed hESC medium and WIBR3 naïve hESCs maintained in 5i/L/A were infected with the lentivirus in the presence of 6 μg/ml polybrene (Millipore). Infected cells were plated on mitomycin C-inactivated DR4 feeder cells and selected with 0.5 μg/ml of ...
Control and miR-139 lentivirus constructs (System Biosciences) were used to generate lentivirus particles with the Lenti-X HTX Packaging System (Clontech) with Lenti-X Concentrator. For in vivo inhibition of miR-139-5p, custom made miRIDIAN Hairpin inhibitors for miR-139-5p or cel-miR-67-3p ...