Fig. S18

Validations of lysosomal biogenesis and acidity in zebrafish embryos. (A) Whole-mount double staining with LysoTracker (10 μM, DND-99; red) and LysoSensor 189 (1 μM, DND-189; green). Live animals at 72 hpf were counterstained with LysoTracker and acidic pH-sensitive LysoSensor 189, simultaneously. LysoSensor 189 weakly detects acidic lysosomal signals in the spns1-mutant animals. Scale bar, 250 μm. Quantification of data presented for LysoSensor 189 (green) and LysoTracker (red) signals in panel A (n = 12) is shown in the right graph; the number (n) of animals is for each genotype. (B) Whole-mount double staining with LysoTracker (10 μM, DND-99; red) and LysoSensor 153 (1 μM, DND-153; green). Animals at 72 hpf were simultaneously counterstained by LysoTracker and neutral pH-sensitive LysoSensor 153. LysoSensor 153 can detect relatively neutral lysosomal signals in the spns1-mutant animals. Scale bar, 250 μm. Quantification of data presented for LysoSensor 153 (green) and LysoTracker (red) signals in panel B (n = 12) is shown in the right graph; the number (n) of animals is for each genotype. (C) Acidic pH-sensitive LysoSensor 189 (1 μM, green) probe in combination with LysoTracker (10 μM, red) was used in wt and spns1-mutant animals, and detectable signals in cells were obtained at 72 hpf. In wt fish treated with pepstatin A and E-64-d (P/E) (5 µg/ml each for 12 h), autolysosomal and/or lysosomal compartments were more prominently detected by LysoSensor 189 at the cellular level with enhanced accumulation of enlarged compartments under the identical LysoTracker staining condition. In contrast, in spns1-mutant animals, the cellular compartments were only weakly detectable by LysoSensor 189. Importantly, the short-term BafA treatment (for 1 h) largely attenuated or abolished staining of acidic compartments by both LysoSensor and LysoTracker, indicating that these autolysosomal and lysosomal compartments in wt animals treated with pepstatin A and E-64-d may retain some strong (lower pH) acidity. Scale bar, 10 μm. Quantification of data presented for LysoSensor 189 (green) and LysoTracker (red) signals in panel C (n = 12) is shown; the number (n) of animals is for each genotype with DMSO, pepstatin A and E-64-d (P/E) and/or BafA (+BafA; 1 h treatment). Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. (D) Using neutral pH-sensitive LysoSensor 153 (green) probes in combination with LysoTracker (red), wt and spns1-mutant animals were examined for detectable signals in cells when stained at 72 hpf. In spns1-mutant animals, autolysosomal and/or lysosomal compartments were more prominently detected by LysoSensor 153 at the cellular level with enhanced accumulation of enlarged compartments. In stark contrast, the cellular compartments in wt fish treated with pepstatin A and E-64-d (P/E) (5 μg/ml each for 12 h) were less detectable by LysoSensor 153 under the same staining condition used with LysoTracker. The short-term BafA treatment (for 1 h) still abolished the acidic compartments stained by both LysoSensor and LysoTracker, suggesting that the autolysosomal and lysosomal compartments observed in spns1-mutants may still retain some weak (higher pH) acidity. Scale bar, 10 µm. Quantification of data presented for LysoSensor 153 (green) and LysoTracker (red) signals in panel D (n = 12) is shown; the number (n) of animals is for each genotype with DMSO, pepstatin A and E-64-d (P/E) and/or BafA (+BafA; 1 h treatment). Three independent areas (periderm or basal epidermal cells above the eye) were selected from individual animals. Error bars represent the mean ± S.D., *p<0.005; ns, not significant in (A), (B) and (D), and **p<0.005; *p<0.05; ns, not significant in (C).