Nature - USA (2019-07-18)

Letter reSeArCH

c

0.5

1.0

1.5

2.0

Lgr5

hi : Paneth

DMSO

GW6471

CHIR99021

0.0090

0.019

0.42

DMSOGW6471 5uMGW6471 5uM

+ Wnt3A 100ng/ml

Isolation of

crypts

4 days in culture

ENR + GW6471 +/- Wnt3A Viability

• 
  

• 
  

• 
  

a

0

-0.1

-0.2

-0.3

Enriched in OLD

Paneth cells

Enriched in YOUNG

Paneth cells

PPARd -SIGNATURE

P-value = 0.042

0.1

-0.1

-0.2

-0.3

-0.4Enriched in OLD Paneth cells

Enriched in YOUNG

Paneth cells

BIOCARTA PPARa PATHWAY

P-value = 0.0073

0.1

0

b

....ATCCGGGACCAGTTCACAGGGCAAGA...

d

Transcription

initiation site

Putative

PPAR

binding site

Notum gene (DECODE)

Exon 12

Stop

codon

Mouse PPARG;RXRA motif (ID: MA0065.2)

Score: 9.61

Relative score: 0.83

3’UTR

Binding motif analysis for PPRE (JASPAR)

0.5

1.0

1.5

Lgr5

hi : Lgr5

lo

2.0

DMSO

GW6471

CHIR99021

0.150.020

0.011

0

5

10

15

20

25

% of epithelial cell

s

Lgr5hi

Lgr5lo

Lgr5-EGFP

Side Scatter

DMSOCHIR GW

0.026

0.048

0

5

10

15

20

% of epithelial cells

DMSOCHIR GW

Paneth 0.0027

Entero-endocrine

Side Scatter

CD24

DMSO

CHIR99021

GW6471

0

2000

4000

6000

8000

Mean CD24 expression (a.u.)

0.0021

0.023

Extended Data Fig. 6 | Decreased PPAR activity in aged intestine.
a, GSEA analysis for ‘Biocarta PPARa’ and ‘PPARd’ gene sets (for statistics,
see ‘RNA sequencing and data processing’ in Methods). Nominal
P value is shown (n =  5 mice in old, n = 4 mice in young). b, Schematic
of the putative PPARα-binding site on the mouse and human NOTUM
genes found with DECODE. Mouse sequence shown. Bottom, score for the
discovered site using JASPAR matrix models for mouse PPAR-response
element (PPRE). PPARG; RXRA motif was used. c, FACS analysis
of cell populations in primary organoids treated for three days
with DMSO, CHIR99021 or GW6471 (n =  6 mice for DMSO, n =  5

mice for CHIR99021 and GW6471). Ratios of Lgr5hi to Paneth cells, and

Lgr5hi to Lgr5lo cells from the same analysis. Mean CD24 expression of

live Epcam+ cells are also shown. d, Representative images of mouse

intestinal organoids treated for 4 days with DMSO, 5 μM GW6471 or

5 μM GW6471 + 100  ng ml−^1 Wnt3A. Arrowheads indicate surviving,

and red asterisks collapsed, organoids. Scale bar, 100  μm. Experiment was

repeated four times with similar results. Unless otherwise indicated, data

are mean ± s.d.; two-tailed unpaired Student’s t-test; exact P values shown

in corresponding panels.