Figure 9.

Mutant W58A-USP14 Increases GABARAP Positive Puncta in a HSC70-dependent Manner

(A and B) Striatal cells were transfected with EGFP-GABARAP in combination with an empty vector or Flag-W58A-USP14 and further stimulated with (A) chloroquine (50 μM, 5 h) or (B) VER-155008 (25 μM, 24 h). Live cell imaging was done using EVOS FL microscope revealing EGFP-GABARAP positive autophagosomes. In (A) right panel represents average integrated intensity per GABARAP particles depicted as a histogram. Intensity of GABARAP positive autophagosomal structures was quantified using ImageJ. n = 3. Scale, 100 μM (A and B).

(C) Striatal cells were plated on Ibidi ibitreat polymer bottom wells as described in Transparent Methods and transfected with EGFP-GABARAP in combination with an empty vector or Flag-W58A-USP14 and stimulated for 24 h with VER-155008 (25 μM). High-resolution live cell imaging with a Nikon-Eclipse Ti-E inverted wide-field microscope equipped with an environmental chamber was utilized to reveal the EGFP-GABARAP positive autophagosomes, n = 5. Scale, 10 μM.

In (A and C) p value was calculated by two-way ANOVA. *p ≤ 0.05, **p ≤ 0.01, ***p ≤ 0.001, ****p ≤ 0.0001. EGFP,- enhanced green fluorescent protein.