Striatal Cells Expressing Mutant Htt Have Reduced Levels of USP14, HSC70, and XBP1u Aggregates
(A) Striatal cells expressing 7Q (control) and 109Q (mutant) huntingtin (Htt) were subjected to immunoblotting for USP14, HSC70, and GAPDH. Lower panels represent histograms of the densitometry ratios of USP14 and HSC70, normalized to GAPDH.
(B) Striatal cells expressing 7Q or 109Q Htt were treated with MG132 (20 μM, 6 h) or DMSO (control) followed by immunoblotting for XBP1u using anti-XBP1 antibody. Right panel represents the histogram for the densitometry ratio of XBP1u normalized to GAPDH.
(C) qPCR was done as described in Transparent Methods. Note reduced expression of USP14 in 109Q Htt cells compared with 7Q Htt cells. Gene expression was normalized to GAPDH and controls set to 1.
(D) Striatal cells were immunostained for XBP1u to show presence of XBP1u-positive ALIS induced by MG132. Note reduced immunostaining in 109Q Htt cells compared with 7Q Htt cells. Right panel represents a histogram for the average integrated intensity per XBP1u particle. Nuclei were stained by Hoechst dye (blue). Scale bar, 100 μm.
In (A–D, except C) n = 3, (C) n = 5, (A–D) p value was calculated by Student's t-test. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001. 7Q, 7-polyglutamine repeats; 109Q, 109-polyglutamine repeats; GAPDH, glyceraldehyde 3-phosphate dehydrogenase.