TUJ1 /DNA

1. Pr1 A Supplementary Figure 1. Characterization of NPC1 knockdown hESC lines. (A) Knockdown of NPC1 measured by qRT-PCR in four clonal NPC1 hESC lines. NPC1 levels were normalized to NONO and HPRT internal controls. Pr= primer set. (B) NPC1 KD hESC can generate cells from the three germ layers through formation of embryoid bodies. Immunofluorescence is shown for ectoderm marker beta-III tubulin (TUJ1), mesoderm marker smooth muscle actin (SMA) and endoderm marker alpha-fetoprotein (AFP). Scale bar is 50μm. (C) NPC1 KD hESC can generate neurospheres and mixed neuronal cultures that express lineage specific markers TUJ1 and MAP2. Immunofluorescence and WB of representative clone are shown. Scale bar is 50μm for neurospheres and 10μm for neuronal cultures. SMA/DNA AFP/DNA TUJ1/DNA C NPC1 KD wt 10 μg 5 μg 10 μg 5 μg NPC1 Pr2 wt NPC1 KD clones 1-4 Pr3 MAP2 phase MAP2/GFP/DNA GFP Pr4 NPC1 level TUJ1 tubulin B

2. wild type wild type U18666A U18666A NPC1 KD NPC1 KD Supplementary Figure 2. Characterization of NPC1 phenotypes in NPC1 knockdown hESC and method for generation of NSC lines. (A) NPC1 KD hESC accumulate LysoTracker positive organelles and internalized HDL particles labeled with Bodipy-cholesteryl. Quantification shown is normalized to wt (** for p<0.001, n=15 from 3 independent experiments). Scale bar is 10um. (B) FACS of hESCs co-cultured with PA6 stromal line. Cultures were dissociated, stained with surface markers and sorted at 12-16 days. CD184+CD15+CD44-CD271- cells were selected, replated and expanded to generate pure NSC lines. NSCs stain positive for lineage markers Ki-67 and Sox2. Scale bar is 50μm. (C) Neural stem cells (NSC) infected with NPC1 shRNA pSicoR-GFP vector have normal morphology and express lineage specific markers nestin and Sox2. Scale bar is 10μm. ** mean LysoTracker intensity C mean Bodipy-HDL intensity ** ** nestin/DNA Sox2/DNA GFP phase A wt U18666A NPC1 KD LYSOTRACKER BODIPY-HDL B **

3. wild type wild type U18666A U18666A NPC1 NPC1 KD Supplementary Figure 3. Additional characterization of mitochondrial phenotypes in NPC1 fibroblasts and neural cells. (A) No significant difference in MitoTracker signal is evident in NPC1 compared to wt fibroblasts grown under baseline conditions, however MitoTracker signal is increased in NPC1 fibroblasts when subjected to serum deprivation. No significant difference of MitoTracker Red-CM-H2XRos staining was observed under baseline or serum deprivation conditions. (B) MitoTracker Red-CM-H2XRos stained NPC1 fibroblasts and neurons do not show mitochondrial fragmentation. Mean mitochondrial length is quantified and normalized to wt (n=10 from 2 independent experiments). Scale bar is 10μm. scramble wt U18666A NPC1 A MITOTRACKER MITOTRACKER RED-CM-H2XROS wt U18666A NPC1 BASELINE BASELINE SERUM DEPRIVED SERUM DEPRIVED B FIBROBLASTS wt U18666A NPC1 mean length (μm) MITOTRACKER RED-CM-H2XROS NEURONS wt U18666A scramble NPC1 KD MITOTRACKER RED-CM-H2XROS mean length (μm)

4. Supplementary Figure 4. Additional characterization of autophagy in NPC1 fibroblasts.(A) Confocal live imaging of human fibroblasts grown under serum deprivation, transfected with GFP-LC3 plasmid and stained with MitoTracker shows co-localization of mitochondrial fragments with LC3-GFP punctae in NPC1 fibroblasts (n=10 from two independent experiments). (B) NPC1 fibroblasts have increased LC3-GFP signal measured by live imaging and flow cytometry. Quantification of LC3-GFP fluorescence intensity is shown (** for p<0.001, * for p<0.05, n=9 from 3 independent experiments). Scale bar is 10μm. wt U18666A NPC1 wild type U18666A NPC1 count A wt U18666A NPC1 LC3-GFP LC3-GFP MITOTRACKER wild type U18666A NPC1 LC3-GFP merge ** * LC3-GFP intensity B