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. 2017 Mar 28;6(1):47–62. doi: 10.3233/JHD-160231

Fig.4.

Fig.4

Autophagy analyses. (A) Western blot analysis of LC3 levels in the microsomal fractions from 6-month-old Htt+/+ (+/+), HttΔQ/ΔQ (ΔQ/ΔQ), HttΔQP/ΔQP (ΔQP/ΔQP), and HttΔN17/ΔN17 (ΔN17/ΔN17) mice. No significant differences were detected in the ratio of LC3-II to LC3-I between different genotypes (HttΔN17/ΔN17: 1.222±0.167, HttΔQP/ΔQP: 1.172±0.154, HttΔQ/ΔQ: 1.204±0.152, mean±SEM, n = 3/genotype; p = 0.6581, 1-way ANOVA). (B-D) Primary striatal neurons isolated from the brains of postnatal day 5 Htt+/+, HttΔQ/ΔQ, HttΔQP/ΔQP, and HttΔN17/ΔN17 mice were grown in serum free media for 8 DIV before treating with 30μM chloroquine (CQ) to assess autophagic flux. (B) Fixed cells were immunostained with antibodies recognizing LC3 (green), p62 (red) and βIII tubulin (blue). Scale bar = 10μm for top 8 panels. Arrowheads in the insets indicate LC3+p62+ cargo-containing autophagosomes within neurites. Scale bar = 5μm for 4 inset panels. (C) Quantification of the LC3+p62+ area within the cell bodies of striatal neurons (p = 0.4350, F = 0.9154, DF = 3, 2-way ANOVA). (D) Quantification of the LC3+p62+ area per μm of neurite in the striatal neurons (p = 0.8792, F = 0.2245, DF = 3, 2-way ANOVA). No significant differences were observed between the Htt+/+ controls and all three domain deletion mutants (n = 40 cells/genotype from 3 different dissections/genotype).