Fig. 2. Gossypol acetate reduced mHTT protein level and neuronal toxicity in the HD cell models.
(A) Gossypol acetate treatment decreased mHTT level in HD patient iPS-derived Q47 neuron cells as shown by HTRF assay (2B7/MW1 for mHTT). The HTRF quantification of HTT level in Q47 cells treated with indicated concentrations of gossypol acetate was shown. Data were normalized to the signal from DMSO group. Statistical analyses were performed by the one-way ANOVA, ***, P<0.001, n=10.
(B) Representative Western-blots showing gossypol acetate’s effect on mHTT levels. Q47 cells were treated with indicated concentrations of gossypol acetate for 24hr. Cell lysate were immunoblotted with HTT antibodies (MW1 for mHTT, or 2B7 for mHTT and wtHTT). β-Tubulin was immunoblotted as a loading control.
(C) Representative Western-blots showing blockage of gossypol acetate’s mHTT-lowering effect of by autophagy inhibitor CQ. Q47 cells were treated with gossypol acetate in the absence or presence of CQ for 24hr. Cell lysate were immunoblotted with the anti-HTT antibodies.
(D) Representative images of Tuj1 immunostaining and caspase 3 activity detection by a green florescent dye. Loss and shrinkage of HD patient iPS-derived neurons (Q47) under the BDNF-deprived condition was shown, which was rescued by mHTT knock-down or gossypol acetate treatment (scale bar: 200 μm).
(E) Quantification of caspase 3 activity in (D) corrected by cell number (by DAPI). Data were normalized to the DMSO control group (first bar). Statistical analyses were performed by one-way ANOVA with post hoc Dunnett’s tests: ***, P<0.001.
(F) Quantification of the Tuj1 signal in (D). Covered area (Tuj1 area) was normalized to the nuclei counts. The Tuj1 area per cell reflects neuronal processes shrinkage and loss. Data were normalized to the DMSO treated control group (first bar). The statistical analysis was performed by one-way ANOVA with post hoc Dunnett’s tests: ***, P<0.001.
For all panels, bars represent mean and SEM