|Add diluted siRNA to diluted Lipofectamine® RNAiMAX Reagent (1:1 ratio).
Incubate for 5 minutes at room temperature and add to cells.
Incubate cells for 1–3 days at 37°C.
One pool containing four siRNAs targeting human AQP5 and a second pool containing four nonspecific siRNAs, as a negative control, were designed and synthesized to have the least off-target effects using SMART-pooled technology (Dharmacon). All siRNA sequences are described in the Supplementary Methods S1. Cells were transfected with 100 nM of siRNAs in Opti-MEM I reduced serum medium (Invitrogen) using RNAi Max transfection reagent (Invitrogen) according to the manufacturer's instruction and cultured at 37°C in a 5% CO2 atmosphere. The medium was removed and replaced with fresh RPMI culture medium supplemented with 10% FBS. The growth of each cell line was measured by MTT assay three days after treatment. Measurements were made in triplicate for each of the cell lines and the experiments were repeated three times. Full paper
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