Interferin hts

Hela cells were grown in clear-bottomed microplates (uClear 384 well microplates, Greiner). Transfection was carried out using Interferin-HTS (Polyplus), according to the manufacturer’s instructions. The siRNA library targeted 840 genes from the Dharmacon Human Druggable siGENOME library, of which 712 were kinase-related, 110 were G-protein coupled receptor-related and 18 were phosphatase-related. After 24 h, cells were accumulated in mitosis for 6 h by addition of 200 nM nocodazole, immunofluorescently stained with mouse anti-H3T3ph (0.1 µg/ml) and rabbit anti-H2BS6ph antibodies (1:1000), followed by fluorophore-conjugated secondary antibodies, and the intensity of staining quantified by High Content Imaging. Experiments were carried out in quadruplicate. The standard score for condition was then calculated, where standard score = (mean staining intensity in presence of siRNA − mean staining intensity for entire plate)/standard deviation of staining intensity for the entire plate; p-values were calculated using one-sample t-test for each siRNA treatment. Approximately 300 cells were measured for each siRNA treatment.

siRNA sequences targeting CRL5 adaptors were purchased from Horizon Discovery and resuspended in nuclease-free water to 1 μM final concentration. A549-κB-LUC cells were reverse-transfected in triplicate replicates with 30 nM siRNA using Interferin-HTS (Polyplus) and incubated for 72 h. The cells were then stimulated with 1 ng/mL of IL-1β for 6 h and subsequently washed with ice-cold PBS and lysed in Passive Lysis Buffer (PLB; Promega). Luciferase activity was measured in a Clariostar plate reader (BMG Biotech) and data for each sample were normalized to its non-stimulated condition and plotted as mean ± SD over the NTC-transfected control. Data shown are representative of 2 independent screens showing similar results. Cells were also reverse-transfected in identical manner to determine cell viability 72 h post-transfection using CellTiter-Glo (Promega) following manufacturer's recommendations.

Cells were transfected in 6-well plates with siRNA using INTERFERin-HTS (Polyplus) transfection reagent in a reverse transfection procedure. Ambion Silencer Select siRNA (20 nMol/L; Life Technologies) was used for all assays. The sense strand sequences for SRP72 were as follows: SRP72 (1): GGACAAGUGUUAUACCGUU and SRP72 (2): GGCAAUUAGUGACCUACAA. Silencer Select Negative Control No. 1 siRNA was used as negative control. The volume of INTERFERin-HTS ranged from 1 µl to 2 µl per well and the seeding density from 150,000 to 200,000 cells per well, depending on the cell line. Cells were re-plated for colony formation assays, knockdown confirmation, and other downstream assays 72 hours after transfection.