Mission lentiexpress human kinases shrna library

The MISSION LentiExpress™ Human Kinases shRNA library (Sigma‐Aldrich, St Louis, MO, USA) was used to screen for candidate protein kinases mediating the growth of PCa cells. Briefly, the AR‐negative DU145 cells were seeded in a 384‐well plate overnight, followed by transduction of lentiviral particles at multiplicities of infection (MOI) of 1 in the presence of 7.5 μg/mL polybrene (Sigma‐Aldrich, St Louis, MO, USA). After 18h incubation, the medium containing the lentivirus particles was replaced with complete medium, and the cell viability was evaluated using the CellTiter‐Glo® assay (Promega, Madison, WI, USA) at 72 hours post‐transduction. Lentiviral particles carrying an empty vector (pLKO.1‐puro), a non‐target shRNA (NS) or a GFP expressing lentiviral construct were included as controls to examine transduction efficiency and well‐to‐well variation. All data were normalized against NS controls and hits were considered when shRNA targeting a specific gene achieved a Z‐score of less than −2.¹², ¹³

The role of protein kinases was examined using the MISSION LentiExpress™ Human Kinases shRNA Library (Sigma, St Louis, MO). Briefly, MDA-MB-468 basal-like breast cancer cells were seeded at a density of 1500 cells/well in a 96-well plate overnight. Cells were transduced with lentiviral particles at multiplicities of infection (MOI) of approximately 1.5 in the presence of 7.5 μg/ml polybrene (Sigma, St Louis, MO) for 18 h in 37°C, 5% CO₂. Medium was changed and the number of viable cells was determined using CellTiterGlo assay (Promega, Madison, WI) 5 days post-transduction. Controls include lentiviral particles carrying an empty vector (pLKO.1-puro), a non-target shRNA (NS) or a GFP-expressing lentiviral construct to monitor transduction efficiency and well-to-well difference. All data were normalized against non-target shRNA controls. Hits were identified based on: 1) at least two independent shRNAs targeting a specific gene exhibit a Z-score of less than −2; and 2) the P-value of Redundant siRNA Activity (RSA) Analysis of each kinase is less than 0.05 [73 (link)].