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 50 mμ . (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 50 m for μneurospheres and 10 m for neuronal cultures.μ

B

TU

J1/D

NA

SM

A/D

NA

AF

P/D

NA

C

phas

e

GF

P

MA

P2/

GF

P/D

NA

NPC1

MAP2

TUJ1

tubulin

wt NPC1 KD5 μg 10 μg 5 μg 10 μg

wt NPC1 KD clones 1-4

A

Pr1

Pr2

Pr3

Pr4

NP

C1

leve

l

0

2

4

6

8

10

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 50 m. μ (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 10 m. μ

LYS

OT

RA

CK

ER

wt U18666A NPC1 KD

BO

DIP

Y-H

DL

0

1

2

3

4

5

****

mea

n Ly

soT

rack

er

inte

nsity

wild type

NPC1 KDU18666A

0

1

2

3

4

5

mea

n B

odip

y-H

DL

in

tens

ity

wild type

NPC1 KDU18666A

****

B

C

phas

e

GF

P

Sox

2/D

NA

nest

in/D

NA

A

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 10 m.μ

MIT

OT

RA

CK

ER

R

ED

-CM

-H2X

RO

S

wt U18666A NPC1

wt U18666A

scramble NPC1 KD

MIT

OT

RA

CK

ER

RE

D-C

M-H

2X

RO

S

0

2

4

6

8

10

mea

n le

ngth

(μ

m)

0

2

4

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8

10

mea

n le

ngth

(μ

m)

wild type

NPC1U18666A

wild type

NPC1 KD

U18666Ascramble

FIBROBLASTS

NEURONS

B

wt U18666A NPC1

Awt U18666A NPC1

MITOTRACKER RED-CM-H2XROSMITOTRACKER

BA

SE

LIN

E

BA

SE

LIN

E

SE

RU

M

DE

PR

IVE

D

SE

RU

M

DE

PR

IVE

D

Bwild type U18666A NPC1

LC3-

GF

P

wtU18666ANPC1

coun

t

LC3-GFP

LC3-

GF

P in

tens

ity

0

1

2

3

***

wild type

NPC1U18666A

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 10 m.μ

ANPC1U18666Awt

LC3-

GF

PM

ITO

TR

AC

KE

Rm

erge