Figure 3. Repeated long-RNA transfection yields sustained, high-level expression of active proteins that modulate downstream targets.

A. Sustaining high levels of Lin28 protein expression by frequent transfection with Lin28-encoding RNA. MRC-5 fibroblasts pre-transfected with a cocktail of siRNAs targeting Ifnb1, Eif2ak2, Stat2, and Tlr3 were transfected five times with 0.5 µg of Lin28-encoding RNA and additional siRNA at 48-hour intervals. Cells were lysed at the indicated times, and the amount of Lin28 protein was analyzed by western blot. β-actin was used as a loading control. B. Sustained expression of Lin28 downregulates its target, mature let7 miRNA. Transfections were conducted as in (A). Data points indicate mature let7a levels in cells transfected once (circles), twice (squares), three times (diamonds), four times (triangles), or five times (crosses), relative to the level in mock-transfected cells. A solid smoothed line connects data points corresponding to cells transfected once, and a dashed smoothed line connects data points corresponding to cells transfected five times (dark symbols). U47 RNA was used as a loading control. Error bars indicate the standard error of replicate samples. C. let7a downregulation is Lin28-specific. Cells were transfected as in (A), but with MyoD1-encoding RNA. Error bars indicate the standard error of replicate samples. D. Expression of MyoD1 protein in fibroblasts. Fibroblasts cultured for three days with or without 2.5 µM 5-aza-dC (AZA) were electroporated with 1 µg/50 µL of MyoD1-encoding RNA. Cells were lysed at the indicated times, and the amount of MyoD1 protein in each sample was analyzed by western blot. E. Expression of MyoD1 in fibroblasts activates its normally silent targets, Cdh15 and Des in a methylation-dependent manner. Cells were transfected as in (D), and expression of Cdh15 and Des was measured by RT-PCR at the indicated times (squares, mock-transfected cells; circles, RNA-transfected cells). F. Regulation of Hmga2 expression by reprogramming proteins. Hmga2 expression in fibroblasts transfected with RNA encoding the indicated protein was measured by RT-PCR 24 hours after transfection. Values are given relative to mock-transfected cells. Gapdh was used as a loading control. *p<0.005. G. Hmga2 expression in fibroblasts co-transfected with RNA encoding reprogramming proteins and a let7a inhibitor was measured by RT-PCR, 24 hours after transfection. Values are given relative to mock-transfected cells that received neither long RNA nor the let7a inhibitor. Gapdh was used as a loading control. *p = 0.004.