Transport, retention, and processing of internalized dsRNA. (A) Trypsin protease treatment traps internalized dsRNA that would otherwise escape during PBS washes. SID-1 expressing S2 cells soaked in increasing concentrations of radiolabeled 500-bp dsRNA were washed extensively in PBS buffer before (PBS) or after (trypsin) trypsin protease treatment (see text). For concentrations higher than 10 ng/mL, increasing amounts of unlabeled 500 bp dsRNA was added and molecules per cell extrapolated. (B) RISC components are required for retention not transport. Five hundred-bp radiolabeled dsRNA was added to Drosophila S2 cells that had been cotransfected with SID-1 expressing plasmids and dsRNA targeting RISC components DCR2 or AGO2 (Supplemental Fig. S1), or luciferase (control). Assays were performed for 30 min and then were washed extensively in PBS buffer before (PBS) or after (trypsin) trypsin protease treatment. The amount of internalized dsRNA was normalized to the control cells cotransfected with luciferase dsRNA. (C) Retained dsRNA is largely unprocessed. 6% polyacrylamide gel electrophoresis of retained radiolabeled 500-bp dsRNA extracted from SID-1 expressing Drosophila S2 cells washed with PBS prior to trypsin treatment, then incubated in fresh media for the indicated hours. siRNA size marker (21 bp) is in the left-most column, siRNA is highlighted in the box. Other dsRNA sizes are indicated on the far left. (D) RISC-dependent retention is energy independent. Five hundred-bp radiolabeled dsRNA was added to Drosophila S2 cells that had been cotransfected with SID-1 expressing plasmids and dsRNA targeting RISC components DCR2 or AGO2 (Supplemental Fig. S1) or luciferase (control). Retention assays with PBS wash prior to trypsin protease treatment were performed at 22°C or 4°C for 30 min and the amount of internalized dsRNA was normalized to the control cells cotransfected with luciferase dsRNA. Error bars = 1 standard deviation; P < 0.005, Student's t-test.
