Profilin is required for the aggregation-inhibitory activity of Y-27632. (A) Control (ctrl), profilin-1 (Prof-1), or profilin-1/profilin-2a-targeting siRNAs were transfected into HEK293 cells, and lysate was evaluated for profilin-1 or profilin-2a by Western blotting. Profilin-1 siRNAs knocked down profilin-1 by 56% and had no effect on profilin-2a; profilin-1/profilin-2a siRNAs knocked down both isoforms by more than 90%. (B) HEK293 cells were transfected with control, profilin-1, or profilin-1/profilin-2a siRNAs along with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP. Relative effects on the aggregation of profilin-1 or profilin-1/profilin-2a siRNAs versus control siRNAs were measured by FRET. Profilin-1 siRNAs had little effect on aggregation; profilin-1/profilin-2a siRNAs increased the aggregation of both Htt exon 1(Q72) (**, *P* < 0.005) and ARN127(Q65) (***, *P* < 0.0001 by unpaired *t* test). Error bars represent the SEM. (C) HEK293 cells were transfected with control, profilin-1, or profilin-1/profilin-2a siRNAs along with Htt exon 1(Q72) CFP/YFP or ARN127(Q65) CFP/YFP with or without Y-27632 (50 μM) treatment. Note that the relative effects of Y-27632 on aggregation in the presence of different siRNAs, not the direct effects of siRNAs on aggregation, as in B, are shown here. Profilin knockdown by both pools of siRNA partially blocked the effect of Y-27632 on ARN127(Q65) aggregation (***, *P* < 0.0005 by unpaired *t* test) and nearly eliminated (profilin-1 siRNAs) or reversed (profilin-1/profilin-2a siRNAs) the effect on Htt exon 1(Q72) aggregation (**, *P* < 0.005; ***, *P* < 0.0005 [unpaired *t* test]). Error bars represent the SEM.

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