A) Confocal analyses of filipin stained WT, nus1 morphant (MO), and ßCD treated nus1 morphant embryos show reduced cholesterol accumulation in the hindbrain, spinal cord (sc), and motor axons of morphant embryos 7dpf. Yellow arrowheads highlight axonal projections motor neurons in ventral spinal cords of nus1 morphants, which are not detected with filipin staining in either WT or ßCD treated embryos. Scale bars =50μM. B) Graph represents quantitation of pixel the average and maximum intensities from hindbrain, spinal cords, and axons of 15 embryos from n=3 experiments. Error=S.E.M, significance calculated by the student’s t-test where ***p<0.001. C) Schematic illustrates workflow for analysis of Lamp1 expression following ßCD treatment. D,E) Western blot analysis and graphic quantitation of Lamp1 abundance (0.5-10h following heat shock induction of its expression) show Lamp1 accumulation is alleviated in nus1 morphants treated with 2.5 mM ßCD. n=4 independent experiments with 25 embryos per sample per experiment. Error=S.E.M, significance calculated by the student’s t-test where *p<0.05, **p<0.01.
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