Knockdown of endogenous DJ-1 leads to decreased DA reuptake.

(A) Western blots for DJ-1 taken from cells that were transfected with either control or 3 different siRNA molecules (siDJ-1#1, siDJ-1#2, siDJ-1#3; Integrated DNA Technologies) were used to induce knockdown of endogenous DJ-1 expressed in HEK-293T cells. Representative immunoblots are shown. (B) The levels of DJ-1 expression was quantified to show significant knockdown of DJ-1 levels in cells transfected with siDJ-1#1 and siDJ-1#3 (50–60% decrease compared to controls), while siDJ-1#2 exhibited a decreasing trend in DJ-1 expression. * P<0.05 vs control group; one-way ANOVA post hoc Tukey test, n = 3. (C and D) DAT function was measure by measuring APP+ uptake levels. Cells were incubated with 20 nM APP+ for 10 min at 37°C, upon which they were imaged with an inverted fluorescent microscope to measure APP+ accumulation. Experiments revealed a significant decrease in APP+ uptake in cells transfected with siDJ-1#1 and siDJ-1#3, while there was decreasing trend in APP+ uptake in cells transfected with siDJ-1#2. * P<0.05, ** P< 0.01 vs control group; one-way ANOVA post hoc Tukey test, n = 3 (8–9 cells from each group was measured in each experiment, for a total of 26 cells per group).