Figure 3.

Aberrant autophagosome and autolysosome formation in tyr^Mut zebrafish embryos. (A) Representative bright-field images showing the pigmentation of 2 dpf zebrafish embryo injected with tyr gRNA+Cas9 protein (tyr^Mut) and their sibling control (CTRL). Scale bar: 0.5 mm. (B) Western blot results showing the level of Lc3-I and Lc3-II proteins in CTRL and tyr^Mut groups treated with chloroquine (CQ). Mean relative ratio of Lc3-II:Tuba was presented under the bands. 50 embryos were collected per group for three independent experiments. Two-way ANOVA with Tukey post hoc was applied, and a significant increase (p < 0.05) of Lc3-I and Lc3-II were detected in tyr^Mut compared with CTRL while no significant differences (p > 0.05) in Lc3-II:Tuba were detected between tyr^Mut treated with CQ and tyr^Mut. (C) Schematic diagram showing the position (periderm or basal epidermal cells above the eye) of imaging. Representative images of nine tyr^Mut Tg(GFP-Lc3) zebrafish embryos stained with LysoTracker from three independent experiments were shown. Three independent areas were selected from individual animals, and the relative number of GFP-Lc3⁺, LysoTracker⁺ and Merged (GFP-Lc3⁺ and LysoTracker⁺) puncta per cell were quantified. Red arrowhead, GFP-Lc3⁺ and LysoTracker⁺ puncta. *, p < 0.05 compared with CTRL. Scale bar: 10 μm (Merged), 5 μm (Enlarged). (D) Schematic diagram showing the position (cells in the midbrain) of imaging. The relative number of GFP-Lc3⁺ and LysoTracker⁻, GFP-Lc3⁻ and LysoTracker⁺, and GFP-Lc3⁺ and LysoTracker⁺ puncta per cell in neurons of midbrain were counted based on Z-Stack (10 layers out of 100 layers) images. Representative images of nine tyr^Mut Tg(GFP-Lc3) zebrafish embryos treated CQ and stained with LysoTracker from three independent experiments were shown. Red arrowhead, GFP-Lc3⁺ and/or LysoTracker⁺ puncta. **, p < 0.01 compared with CTRL; ^##, p < 0.01 compared with tyr^Mut. Scale bar: 40 μm (Merged), 3 μm (Enlarged)