Fig. 4.

RNAi knockdown of trafficking proteins in S2 cells. Hrs and Rab5 dsRNA treatment of S2R+ cells. These cells endogenously express DFz2. Rab5 dsRNA treatment stimulates Wg-induced activation of the TOP-Flash reporter compared to control DsRed dsRNA treatment. Knockdown of Axin, a negative regulator of the pathway, serves as a positive control. This figure reports the averages of triplicates from one representative trial. In this particular experiment, a Student’s t test showed a statistically significant difference between the average luciferase value of Rab5 dsRNA-treated cells and the control DSRed dsRNA value (P < 0.001). Knockdown of Rab5 was performed in five separate experiments. In one experiment (not shown), extra samples were measured in order to compare 3 and 5 days post-transfection luciferase levels. Luciferase values for the 3-day Rab5 knockdown samples were not significantly different from controls, whereas the 5-day samples were different. Rab5 knockdowns had significantly higher luciferase reporter levels than controls in the other four experiments (all assayed at 5 days). The fold increase over mock-treated cells ranged from 1.6 to 2.3. The Rab5 dsRNA results were dependent on Wg production (data not shown). All four experiments testing dsRNAs directed toward Hrs were negative (including two trials that incorporated two dsRNAs, with each tested individually and in combination).