A B iPSC - CTL1 iPSC - CTL2 iPSC - CTL2 · 2020. 2. 18. · of 4 technical replicates. CTG:...

0 103102 104 105

A BiPSC - CTL1 iPSC - CTL2

iPSC - LND1 iPSC - LND2

SSEA

3

105

104

103

102

0

iPSC - CTL1

0 103102 104 105

TRA-1-81

iPSC - CTL2105

104

103

102

0

0 103102 104 105

iPSC - LND1

105

104

103

102

0

iPSC - LND2

105

104

103

102

0

0 103102 104 105

Figure S1 - Ruillier et al- Revision

SSEA

3

TRA-1-81

SSEA

3

TRA-1-81

SSEA

3

TRA-1-81

iPSC - CTL1

iPSC - LND2

iPSC - CTL2

iPSC - LND1

Hoe

chst

/SSE

A-4/

Oct

-4

C

Figure S1: CTL and LND donor-derived iPSC

A - Bright field microscopy of colonies with typical iPSC morphology. Control HGPRT

competent cells: CTL1 and CTL2. Lesch-Nyhan disease HGPRT- deficient cells: LND1 and

LND2. Scale bar = 200 μm. B- Representative images of self-renewing HGPRT competent

(CTL1 and CTL2) and deficient (LND1 and LND2) iPSC lines expressing the 2 pluripotency

markers SSEA-4 (green) and Oct-4 (red) in colonies. Scale bar = 200 μm. C - Flow cytometry

analysis of SSEA3 and TRA-1-81 pluripotency markers.

B

HG

PRT

activ

ity

(nm

ol N

ADH

/h/m

g of

pro

tein

)

0

2

4

6

CTL1

CTL2

LND1

LND2

iPSC

CTL1

CTL2

LND1

LND2

CTL1

CTL2

LND1

LND2

NSC Neurons

A#1

Con

trols

LNDHoe

chst

/Nes

tin/S

ox2

#2


CTL1 CTL2 LND1 LND2C

Hoe

chst

/CU

X1/T

uj-1

Hoe

chst

/Tuj

-1/B

rn2

Figure S2: Phenotypical characterization of neurons differentiated from CTL and LND

stem cells

A - Representative images of neural stem cells (NSC) expressing Nestin (green) and Sox2 (red).

Scale bar = 200 μm. B - HGPRT enzymatic activity in CTL and LND-derived iPSC, NSC and

neurons. The results are expressed as nmol of NADH produced per hour and normalized to total

protein content. C – Immunostaining of the cortical superficial layer markers CUX1 and Brn2

in Tuj-1 positive neurons after 18 days of NSC differentiation. Scale bar: CUX1 = 25 μm, Brn2

= 50 μm.

Hypoxantine

Xanthine

Uric acid

IMP

InosineGuanosine Adenosine

AMPGMP

Guanine

GTP ATP

HGPRT HGPRT

Salv

age

path

way

Deg

rada

tion

De

novo

syn

thes

is p

athw

ay

PRA

PRPP

PPATGln

FGAM

FGAR

PFASGln

AZA

AZA


B

Hyp

oxan

thin

e (μ

mol

/l)

0

20

40

60

80

100

CTLs

LNDs

A

0

20

40

60

80

100

Aza: 0.5 μM 2μM vGluts GABA vGluts GABA

% o

f neu

ron

sub-

type

s / D

MSO

LNDCTL

Hoe

chst

/vG

LUTs

/H

uC/D

Hoe

chst

/G

ABA

C DCTL1 CTL2 LND1 LND2

Figure S3: Inhibition of de novo synthesis using asazerine

A - Quantification of hypoxanthine synthesis in CTL and LND cells. B-. Inhibition of purine

de novo synthesis by Azaserine. C- Immunostaining of glutamatergic and GABAergic neuronal

sub-types in CTL and LND cells treated with DMSO 0.1% or azaserine 2 μM from day 5 of

differentiation. Scale bar = 25μm. D- Quantification of the different neuronal sub-types after

DMSO or azaserine treatment. Results are expressed as mean +/- SD of 2 cell lines in 3

biological replicate. C-D: The analysis of neuronal subtypes were performed at day 18 of

differentiation.

100

150

50

0-7 -6 -5 -4

EC50 = 0.52 μM

100

150

50

0% o

f rec

over

y (H

oech

st)

Log10 [M]

% of viability (H

oechst)

N6-MA

-7 -6 -5 -4

EC50 = 12.9 μM

% o

f rec

over

y (H

oech

st)

Log10 [M]

% of viability (H

oechst)

Thio-NADP

100

150

50

0

100

150

50

0

-7 -6 -5 -4

EC50 = 9.74 μM

% o

f rec

over

y (H

oech

st)

Log10 [M]

% of viability (H

oechst)

NADPH

100

150

50

0

100

150

50

0

-7 -6 -5 -4

EC50 = 6.52 μM

% o

f rec

over

y (H

oech

st)

Log10 [M]

% of viability (H

oechst)

SAM

100

150

50

0

100

150

50

0

-7 -6 -5 -4

EC50 = 18.5 μM

% o

f rec

over

y (H

oech

st)

Log10 [M]

% of viability (H

oechst)

NAD+

100

150

50

0

100

150

50

0

-7 -6 -5 -4

EC50 = 7.01 μM

% o

f rec

over

y (H

oech

st)

Log10 [M]

% of viability (H

oechst)

Ap4A

100

150

50

0

100

150

50

0


Figure S4: Dose-response analysis of the 6 hit compounds efficiency using Hoechst

staining as a read-out.

The black curve corresponds to the percentage of recovery after treatment of LND1 NSC with

1.0 μM azaserine. The blue line shows the percentage of viability after treatment with the drug

alone without azaserine, representing the toxicity of the compound itself. Results are expressed

as mean ± SD of 4 technical replicates (2 biological replicates).

Non ed

ited 1

CRISPR-1

CRISPR-2

Non ed

ited 2

A

HGPRT

β-Actin

Non ed

ited 1

Non ed

ited 2

CRISPR-1

CRISPR-2

B

0

2

4

6

Non edited 1Non edited 2CRISPR-1CRISPR-2

C

HG

PRT

activ

ity

(nm

ol N

ADH

/h/m

g of

pro

tein

)

100

150

50

0-8 -7 -6 -5

% o

f via

bilit

y (C

TG)


Azaserine

Log10 [M]

D

% o

f via

bilit

y (C

TG)

E

CTL1CTL2LND1LND2

100

150

50

0-3 -2 -1 0

CTL1CTL2LND1LND2

% o

f via

bilit

y (C

TG)

100

150

50

0-9 -8 -7 -6

HATLog10 [X]

RotenoneLog10 [M]

Figure S5: Primary hits compounds specificity.

A- Western-blot analysis of HGPRT protein expression in CRISPR/Cas-9 edited NSC-derived

from the human embryonic stem cells line SA001 (CTL3). Two non-edited clones and 2 clones

invalidated for HGPRT (CRISPR-1 and CRISPR-2) were compared. β-actin was used as a

loading control. B - Enzymatic HGPRT activity measurement in the same clones. C – Azaserine

selective toxic effect upon HGPRT-edited CRISPR NSC. Results are expressed as mean ± SD

of 4 technical replicates. CTG: Cell-TiterGlo. D- CTG estimation of cell viability after

treatment of CTL and LND cell lines with increasing concentrations of HAT medium

(Hypoxanthine, Aminopterin and Thymidine containing medium). The X axis represents HAT

medium concentration express as log10 (X), where the X is the dilution factor of the commercial

HAT complete medium. Results are expressed as mean ± SD of 4 technical replicates and 2

biological replicates. E- CTG estimation of cell viability after treatment of CTL and LND cell

lines with increasing concentrations of rotenone on CTL and LND NSC lines. X axis represents

compound concentration expressed as log10 (Rotenone concentration in molar M). Results are

expressed as mean ± SD of 4 technical replicates and 2 biological replicates.

LND1Azaserine + DMSO


Hoe

chst

/GAB

AH

oech

st/G

ABA

Hoe

chst

/vG

luts

Hoe

chst

/vG

luts

Azaserine + SAM

Azaserine + DMSO Azaserine + SAMLND2

Figure S6: Analysis of neuronal subtypes rescued by SAM after azaserine treatment of

LND cells.

GABA: GABAergic neurons, vGluts: Glutamatergic neurons, SAM : S-adenosylmethionine.

Scale bar = 50 μm.

Table S1: List of CTL and LND hiPSC or hESC lines

Study code

Cell type

Donnor code

Cell of origin Provider Karyotype HGPRT mutation Clinical

description

CTL1 hiPSC GM01869 fibroblasts Coriell, USA 46, XY no unaffected

CTL2 hiPSC GM04603 fibroblasts Coriell, USA 46, XY no unaffected

CTL3 hESC SA001 Blastocyst Cellartis, Sweden 46, XY no unaffected

LND1 hiPSC GM02227 fibroblasts Coriell, USA 46, XY inv/del, ex6‐9

Edwards et al, 1989 LND

LND2 hiPSC GM23784 fibroblasts Coriell, USA 46, XY IVS7 + 5 G>A

Gibbs et al, 1990 LND

Table S2: List of primary antibodies, providers and dilutions.

Antibody Host Reference Provider Dilution HPRT1 Rabbit 15059‐1‐AP Proteintech, Rosemont, IL, USA 1:500

PRTFDC1 Rabbit 11986‐1‐AP Proteintech, Rosemont, IL, USA 1:250

SSEA‐4 Mouse 4755S Cell Signaling, Danvers, MA, USA 1 :500 Oct‐4 Rabbit 2840S Cell Signaling, Danvers, MA, USA 1 :500 Nestin Mouse MAB5326 Merck, Darmstadt, Germany 1 :500 Sox2 Rabbit AB5603 Merck, Darmstadt, Germany 1:500 Ki‐67 Mouse MAB4190 Merck, Darmstadt, Germany 1:500 HuC/D Mouse A‐21271 Thermo Scientific, Waltham, MA, USA 1:250

Tubulin β‐3 (Tuj‐1) Rabbit 802001 Biolegend, San Diego, CA, USA 1:1000

TRA‐1‐81 AF647 Mouse 330706 Biolegend, San Diego, CA, USA 2 µg/ml SSEA‐3 PE Rat 330312 Biolegend, San Diego, CA, USA 16 µg/ml

Table S3: Table summarizing the 32 primary hits from LOPAC, Prestwick and SelleckChem libraries.

Library Conc. CAS Compound name % Recovery Z‐Score Run Z‐Score Plate

LOPAC 10 µM 102783‐36‐8 P1,P4‐Di(adenosine‐5’) tetraphosphate triammonium 181.99 39.97 16.68 LOPAC 10 µM 19254‐05‐8 Thio‐NADP sodium 65.68 14.78 12.87 LOPAC 10 µM 1867‐73‐8 N6‐Methyladenosine 64.55 14.53 10.39 LOPAC 10 µM 2646‐71‐1 NADPH tetrasodium 59.48 13.43 9.6

Prestwick 5 µM 53‐84‐9 Nadide 17.11 4.26 6.06 Prestwick 5 µM 66‐81‐9 Cycloheximide 13.55 3.49 4.93 LOPAC 10 µM 85666‐17‐7 Furegrelate sodium 12.45 3.25 1.29

Prestwick 5 µM 5536‐17‐4 Vidarabine 12.30 3.22 4.53 Prestwick 5 µM 1867‐73‐8 N6‐Methyladenosine 11.29 3.00 4.98 LOPAC 10 µM 130506‐22‐8 6‐Nitroso‐1,2‐benzopyrone 9.86 2.69 1.85

Prestwick 5 µM 21679‐14‐1 Fludarabine 9.81 2.68 5.62 LOPAC 10 µM 134523‐03‐8 Atorvastatin calcium salt trihydrate 9.71 2.66 3.42 LOPAC 10 µM 38819‐10‐2 Ara‐G hydrate 7.46 2.17 1.91 LOPAC 10 µM 501‐36‐0 Resveratrol 7.48 2.17 1.91

Prestwick 5 µM 73573‐88‐3 Mevastatin 6.78 2.02 3.29

PW Phyto 10 µM 6027‐98‐1 Harmaline hydrochloride dihydrate 100.55 8.88 8.88

SelleckChem 5 µM 827022‐32‐2 PD‐0332991 (Palbociclib) HCl 20.00 6.57 6.08 SelleckChem 5 µM 827022‐33‐3 PD0332991 (Palbociclib) Isethionate 18.66 6.17 7.21 SelleckChem 5 µM 1144068‐46‐1 WYE‐125132 14.73 5.01 4.83 SelleckChem 5 µM 1009298‐09‐2 AZD8055 13.72 4.70 5.48 SelleckChem 5 µM 741713‐40‐6 R547 10.40 3.72 3.57 SelleckChem 5 µM 1207360‐89‐1 GDC‐0349 10.09 3.63 3.41 SelleckChem 5 µM 343787‐29‐1 CP‐673451 9.9 3.57 3.36 SelleckChem 5 µM 1009298‐59‐2 AZD2014 8.56 3.17 3.67 SelleckChem 5 µM 147526‐32‐7 Pitavastatin calcium (Livalo) 8.35 3.11 3.59 SelleckChem 5 µM 1056634‐68‐4 Momelotinib (CYT387) 5.81 2.36 2.25 SelleckChem 5 µM 670220‐88‐9 Crenolanib (CP‐868596) 5.56 2.29 2.17 SelleckChem 5 µM 936890‐98‐1 OSI‐027 5.52 2.27 2.17 SelleckChem 5 µM 1224844‐38‐5 INK 128 (MLN0128) 5.46 2.25 2.58 SelleckChem 5 µM 1223001‐51‐1 Torin 2 5.46 2.25 2.58 SelleckChem 5 µM 928037‐13‐2 Golvatinib (E7050) 5.20 2.18 2.49

SelleckChem 5 µM 50357‐45‐4 Pentamidine HCl 5.04 2.13 2.43

A B iPSC - CTL1 iPSC - CTL2 iPSC - CTL2 · 2020. 2. 18. · of 4 technical replicates. CTG:...

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