Cellular Assays of Viability, Apoptosis, and Gene Expression

Measurements of cell viability were performed after transfection of cells with the siRNA and/or treatment with recombinant or native proteins using the MTT assay (LONZA, Mississauga, Canada). Briefly, when recombinant (LIF and IL6) or native proteins (C1q and anti-IL6) were used, at the listed concentrations, these were added at 72 hours after transfection with siRNA followed by end point assays of viability (N = 3) or gene expression (N = 3) at 96 hours. In viability assays using siRNA-mediated gene inhibition, 10 μL of MTT reagent (30-1010K; ATCC) was added to each well 93 hours after transfection using a multichannel repetman (Thermo Scientific), and then incubated for 3 hours at 37°C. Detergent reagent (100 μL, 30-1010K; ATCC) was then added using a multichannel repetman (Rainin, Mississauga, Canada). Plates then were covered with tin foil to protect them from light and left at room temperature overnight. Cell viability was calculated by reading the absorbance at 570 nm using a spectrophotometer (Tecan Spark 10 M, Männedorf, Switzerland). In experiments using fostamatinib R406, cells were treated with R406 or vehicle dimethyl sulfoxide at the listed concentration for 24 hours followed be measurement of cell viability. Cellular growth arrest and apoptosis after R406 treatment was determined through flow-cytometric cell-cycle analysis using fluorescein isothiocyanate–labeled bromodeoxyuridine incorporation and a 7-AAD staining kit (BD Biosciences, San Jose, CA), as per the manufacturer’s instructions, and analyzed using a BD FACSVerse (BD Biosciences, San Jose, CA). Cells undergoing growth arrest were defined by a decreased percentage of cells in the S phase (R4) and a gain of those in G0/G1 (R3). Cells undergoing apoptosis were defined by a decreased percentage of cells in the S phase and a gain in both G0/G1 (R3) and sub-G1 populations (R6). Controls including deoxycholate treatment (200 μmol/L) and cells in culture media lacking FCS (resting-FCS) were used to define cell populations in apoptosis and growth arrest, respectively.