GENE EXPRESSION PROFILING OF CUTANEOUS ...

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GENE EXPRESSION PROFILING OF CUTANEOUS INJURED AND NON-INJURED ... (A) Analysis of transcript levels for known somatosensory mediators (list modified ... Scatter plots of top 5% genes on the microarrays for the sham condition ...
GENE EXPRESSION PROFILING OF CUTANEOUS INJURED AND NON-INJURED NOCICEPTORS IN SNI ANIMAL MODEL OF NEUROPATHIC PAIN Temugin Berta, Florence E. Perrin, Marie Pertin, Raquel Tonello, Yen-Chin Liu, Alex Chamessian, Ann C. Kato, Ru-Rong Ji, and Isabelle Decosterd

Supplementary Figures 1-3

A

B

C

Peripherin

NF200

Merge 100 mm

2000 1600 1200 800 400 0

E

NF200

PRPH

F

Peripherin NF200

50 2000 1600 1200 800

Frequency (%)

Peripherin

Cross sectional area (mm2)

Cross sectional area (mm2)

D

40

Cut off area at 500 mm2

30 20 10

400 0 0

0-5 1

5-10 2 10-15 3 15-20 4 20-25 5 25-30 6 30-35 7 35-40 8

Small Medium

Large

Cross sectional area (102mm2)

Supplementary Fig. 1: Definition of the cell size criteria for the LCM of nociceptors. Immunohistochemistry of fresh DRG tissue for (A) nociceptor marker peripherin, (B) large-sized neuron marker NF-200 and (C) merged figures (scale bars = 100 mm). Analysis of average cross-sectional area for (D) peripherin and (E) NF-200 in L4-L5 sections issued from 1 week SNI or sham animals. To note no significant differences were detected between the two surgeries. (F) Analysis of the cross-sectional area distribution for the peripherin- and NF-200-positive neurons reveal an optimal cut off cross-sectional area of 500 mm2, within 90% of neurons were peripherin positive and only 5% were NF-200 positive.

B

A P A A A

Pvalb Runx3 Etv1 Etv4 Cdh12 Slc17a7

A A P A A A

Ntrk1 Ntrk2 Ntrk3 Gfra1 Gfra2 Gfra3 Ret

P P P P P P P

Chip2

Fam38b Trpc1 Slc17a8 Th Mrgprb4

C h ip 1

P P A P A P P P P A P A P P P

6000

Ret

4000

2000

P2rx3

Runx1 0 0

5000

10000

15000

20000

C h ip 2

Chip1 TrkA

Calca

8000

Chip 3

Trpv1 Trpv2 Trpa1 Trpm3 Trpm8 Scn9a Scn10a Scn11a P2rx3 Mrgprd Runx1 Tlx3 Calca Calcb Tac1

Scn11a

Scn10a Trpv1

8000

C h ip 1

Present/ Absent

Scn10a Trpv1 P2rx3

6000

Scn11a

4000

Ret 2000

Calcb Runx1

0 0

2000

4000

6000

8000

10000

C h ip 3

Chip 2 TrkA

Calca

8000

Chip 3

Neurotrphic receptors Proprioception Tactile function

Thermosensation/Nociception

Gene

10000

C h ip 1

A

Calca

TrkA

Scn10a Trpv1

6000

Scn11a

4000

Ret P2rx3

2000

Calcb Runx1

0 0

5000

10000

15000

20000

C h ip 3

Chip 1

Supplementary Fig. 2: Expression of somatosensation markers on microarray analysis from LCM samples. (A) Analysis of transcript levels for known somatosensory mediators (list modified from Chiu et al. (ref)) in LCM samples from sham condition shows the presence of several markers for thermosensation and nociception. (B) Scatter plots of top 5% genes on the microarrays for the sham condition reveal the high expression of typical nociceptive markers, such as Calca (CGRP), Scn11a (Nav1.9) and Trpv1.

Up-regulated genes Fold >2 >5

Non-injured 15 3

Injured 422 91

> 10

0

33

Down-regulated genes Fold >2 >5 > 10

Non-injured 6 0 0

Injured 180 7 0

Supplementary Fig. 3: Fold change of regulated gene. Number of up-regulated and down-regulated genes per conditions compared to sham and classified by fold change (for statistic see method section).