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