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. 2010 Jul 23;5(7):e11756. doi: 10.1371/journal.pone.0011756

Figure 2. Innate immune suppression enables frequent long-RNA transfection.

Figure 2

Combinatorial siRNA screening identifies siRNA cocktails that rescue cells from the innate immune response triggered by long-RNA transfection. A. Upregulation of innate immune genes following long-RNA transfection. MRC-5 fibroblasts were transfected with 0.4 µg of RNA per well of a 24-well plate using lipids. Expression of innate immune genes was measured by quantitative RT-PCR 24 hours after transfection. Gapdh was used as a loading control. Error bars indicate the standard deviation of replicate samples. B. Repeated long-RNA transfection causes cell death in human fibroblasts. MRC-5 fibroblasts were electroporated twice with 0.5 µg/50 µL of Lin28-encoding RNA at 48-hour intervals. Samples of cells transfected with RNA (black circles) and mock-transfected cells (gray squares) were trypsinized and counted at the indicated times. Data points and error bars indicate the mean and standard error of two independent experiments. Data points are connected for clarity. C. Combined knockdown of Ifnb1, Eif2ak2, and Stat2 rescues cells from the innate immune response triggered by frequent long-RNA transfection. MRC-5 fibroblasts were transfected as in (B), but with the indicated siRNA on day 0, and 0.5 µg of Lin28-encoding RNA and additional siRNA on days 2 and 4 (Table S1). Samples of cells were trypsinized and counted 24 hours after the second long-RNA transfection (day 5). Values indicate cell count relative to mock-transfected cells. †Standard error of replicate samples (n = 4). *p<0.05, **p<0.005. D. Frequent transfection of primary human fibroblasts with a mixture of RNA encoding the reprogramming proteins Oct4, Sox2, Klf4, and Utf1 yields sustained, ES-cell-level expression. Cells were reverse transfected with an immunosuppressive siRNA cocktail (Lipofectamine RNAiMAX, Invitrogen), and then transfected with protein-encoding RNA (0.1 µg of RNA per factor per well) using lipids every day for three days. Cells were fixed and stained 8 hours after the last transfection. For each protein, identical camera settings and exposure times were used for the mock-transfected, RNA-transfected, and hES-cell samples. E. Cells expressing reprogramming proteins undergo mitosis. Cells were transfected as in (D). Utf1 localized to chromosomes in mitotic cells (arrow).