Figure 7

ITCH rescues polyglutamine aggregation and cytotoxicity.

(A–C) Plasmids encoding wild-type ITCH and mutant ITCH-C822S were transiently cotransfected with constructs expressing expanded-polyglutamine huntingtin (EGFP-HDQ74) into Cos-7 cells. After 24 h, the cell extracts were immunoblotted with anti-GFP and anti-actin antibodies (A). Similar experiments were performed with expanded-polyglutamine ataxin-3(Q84) and the blots were probed with anti-GFP and anti-actin antibodies (B). Cells that coexpressed wild-type ITCH with EGFP-HDQ74 and ataxin-3(Q84) proteins were treated with 10 μM MG132 and 50 nM bafilomycin (Baf). The cell lysates were used for immunoblotting with anti-GFP and anti-actin antibodies (C). Quantification of band intensities collected from three different experiments as shown in section A–C using NIH image analysis software. *, P < 0.05 compared with the control. (D–F) ITCH stimulates the degradation of misfolded heat-denatured luciferase. Cos-7 cells were transiently transfected with normal ITCH (D) and mutant ITCH-C822S (E) plasmids together with the firefly luciferase expression construct. After 48 h of transfection, the cells were processed for luciferase activity and immunoblotting with anti-luciferase and anti-actin antibodies was performed (F). Band intensities were quantified by using NIH image analysis software. *, P < 0.05 compared with the control. (G) Cells were transfected with ITCH plasmid as shown in figure, after 48 h, aggregation and quantitation was monitored for EGFP-HDQ74 under a fluorescence microscope. (H) 293T cells were cotransfected with mutant-ITCH-C822S as shown in figure, after 48 h, aggregation was monitored under a fluorescence microscope. Quantitation of EGFP-HDQ74 aggregates in cells transfected with mutant-ITCH-C822S plasmid and ITCH-siRNA oligonucleotides. The results are presented ± S.D. of three independent experiments each performed in triplicate. *, P < 0.05 compared with the control siRNA. (I) Immunoblot analysis of Cos-7 cells transfected with pcDNA and wild-type Myc-ITCH constructs. 293T cells were transiently transfected with control (scrambled siRNA) and ITCH-siRNA oligonucleotides. Cell lysates were prepared and immunoblotted with anti-ITCH and anti-actin antibodies. Full-length blots are presented in Supplementary Figure-S7 (J) Assessment of cytotoxicity mediated by expanded-polyglutamine EGFP-HDQ74 proteins cotransfected with wild-type ITCH or mutant-ITCH-C822S constructs, or ITCH-siRNA oligonucleotides. Cell viability was measured using the MTT assay. Values are the mean ± S.D. of three independent experiments, each performed in triplicate. *, P < 0.05 compared with the control (control siRNA and empty pcDNA) sets.