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. 2003 Jul 22;23(13):3813–3826. doi: 10.1016/j.celrep.2018.05.098

Figure 5.

Figure 5

GM2 Accumulation in Lysosomes Promotes the Accumulation of Autophagy Marker p62 in Lysosomes

(A) Proportion of lysosomes stained with Lamp1 that were also positive for p62 in Spg11+/+ and Spg11−/− neurons after 3 and 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗p = 0.04 and ∗∗∗p < 0.001.

(B) Immunostaining of Spg11−/− neurons with GM2 antibody (cyan), lysosomal marker Lamp1 (magenta), and the autophagy marker p62 (yellow). Arrowheads indicate lysosomes, which were positive for p62 and GM2 staining. Scale bar: 10 μm.

(C) Proportion of lysosomes stained with Lamp1 that were also positive for both p62 and GM2 in Spg11+/+ and Spg11−/− neurons after 3 and 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001.

(D) Effect of miglustat treatment (100 μM) on the proportion of lysosomes stained with Lamp1 that were also positive for p62 in Spg11+/+ and Spg11−/− neurons after 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001.

(E) Effect of the downregulation of GM3 synthase with two independent miRNAs (GM3S-1 and GM3S-2) on the proportion of lysosomes stained with Lamp1 that were also positive for p62 in control neurons after 6 days in vitro. The graph shows the mean ± SEM values. Two-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001.

(F) Effect of Neu1 downregulation with two independent miRNAs (Neu1-1 and Neu1-2) on the proportion of lysosomes stained with Lamp1 that were also positive for p62 in control neurons after 6 days in vitro. The graph shows the mean ± SEM values. One-way ANOVA, followed by Holm-Sidak post hoc test; ∗∗∗p < 0.001.

In (A–D), day in vitro 3 (DIV3), N = 47–50 neurons quantified in two independent neuron preparations; DIV6, N = 173–193 neurons quantified in four independent neuron preparations. In (E), N = 7–29 neurons quantified in two independent neuron preparations. In (F), N = 57–68 neurons quantified in three independent neuron preparations.