Supplementary Table A1. List of siRNAs used in this study

Name Distributor Sequence

       

siCtrlAllstars Negative Control siRNA

Qiagen, Venlo, The Netherlands

seq not provided

Rat siPTPN2 #1

ON-TARGETplus rat PTPN2

SMARTpool®

Thermo Scientific,

Chicago, IL, USA

seq #1 5’-GUAGAGAAAGAAUCGGUUA-3’ seq #2 5’-GAAACAGGAUUCAGCGUGA-3’ seq #3 5’-UGAUCACAGUCGUGUUAAA-3’

seq #4 5’-CUACAUGGCCAUAAUAGAA-3’

Rat siPTPN2 #2

Silencer® Select Pre-designed

siRNA rat PTPN2

Applied Biosystems, Austin, TX,

USA

5’-GCAUUCGAGAGGAUAGAAA-3’

Human siPTPN2 #1

Silencer® Select Validated siRNA human PTPN2 #1

" 5’-GGAGAUUCUAGUAUACAGA-3’

Human siPTPN2 #2

Silencer® Select Validated siRNA human PTPN2 #2

" 5’-GUACAGGACUUUCCUCUAA-3’

Rat siSTAT1BLOCK-iT

Stealth™ Select siRNA

Invitrogen, Paisley, UK

5’-CCCUAGAAGACUUACAAGAUGAAUA-3’

  Std   real time PCR

  Sequence (5’- 3’)

(bp)  Sequence (5’- 3’)

(bp)

Rat GAPDHF ATGACTCTACCCACGGCAAG

975F AGTTCAACGGCACAGTCAAG

118R TGTGAGGGAGATGCTCAGTG R TACTCAGCACCAGCATCACC

Human GAPDHF CTGAGAACGGGAAGCTTGTC

555F CAGCCTCAAGATCATCAGCAA

106R AGGTCAGGTCCACCACTGAC R TGTGGTCATGAGTCCTTCCA

Rat -actinF ATGGTGGGTATGGGTCAGAA

918F CTGTGCCCATCTATGAGGGT

133R CAGTGAGGCCAGGATAGAGC R CTCTCAGCTGTGGTGGTGAA

Rat srp-14F AGACCAGGATGGTGTTGCTC

436F CGTGTTCATCACCCTGAAGA

109R TAGACCCTTCCGTGAAAACG R CGTGGCCCTTAACAGACACT

Mouse/rat/human PTPN2

F TGTCCGCTGTGGTAGTTCC328

F ATCGAGCGGGAGTTCGA110

R AATGGCAGCATGTGTTCG R TCTGGAAACTTGGCCACTC

Mouse/rat/human PTPN22

F ATTTGACATGCCCTCCCT398

F GGCATGGACCAAAGAGAAAT102

R ATCATCCTCCAGAAGTCCAG R CCTTTTCAGCTTCAGAAATTCA

Rat Insulin 1F CATCAGCAAGCAGGTCATTG

316F ACCTTTGTGGTCCTCACCTG

118R TGCAGCACTGATCCACAATG R AGCTCCAGTTGTGGCACTTG

Rat Insulin 2F TGACCAGCTACAGTCGGAAA

390F TGTGGTTCTCACTTGGTGGA

108R GTTGCAGTAGTTCTCCAGTTGG R CTCCAGTTGTGCCACTTGTG

Supplementary Table A2. List of primers used for real time PCR

Laemmli Buffer25 mM Tris-HCl pH 6.8, 10 % glycerol, 1 % SDS, 350 mM 2-mercaptoethanol, 175 mM dithiothreitol, 0.005 % Bromophenol Blue

Phospho Lysis Buffer

1% NP40, 25 mM Hepes pH 7.8, 150 mM NaCl, 1 mM CaCl2, 1 mM MgCl2, 50 mM NaF, 1 mM

Na3VO4, 1 mM PMSF

Supplementary Table A3. Lysis buffers used for Western blot

Supplementary Table A4. List of antibodies used for Western blot analysis

Antibody Company Reference Dilution

       

PTPN2 R&D Systems, Abingdon, UK

clone 252294

1:1000

STAT1 Santa Cruz Biotechnology, Santa Cruz, CA.,U.S.A.

sc-346 1:1000

Insulin R sc-711 1:1000

phospho-STAT1 (Y701)

Cell Signaling, Danvers, MA, USA

#9171 1:1000

phospho-STAT3 (Y705) #9131 1:1000

STAT3 #9132 1:1000

phospho-p44/42 MAPK #4377 1:1000

p44/42 MAPK #9102 1:1000

phospho-EGFR #2236 1:1000

EGFR #2232 1:1000

phospho-Insulin R (pYpY1162/1163)Biosource, Camarillo, CA, USA

44-804G 1:1000

-tubulinSigma, Bornem, Belgium

T9026 1:5000

HRP-conjugated anti-rabbit IgG Lucron Bioproducts, De Pinte, Belgium

711-036-152

1:5000

HRP-conjugated anti-mouse IgG715-036-

1501:5000

0

1

2

3

Fol

d in

duct

ion

CtrlIL-1IL-1 + IFN

*

GAPDH -actin srp-14

PT

PN

22 /

GA

PD

H

0,00

0,05

0,10

0,15

0,20

200,00

300,00

NSIL + IFN

INS-1E cells

Primary rat -cells

Human islets

Spleen Lymph nodes

200

300

Supplementary Figure A1. Comparison between the expression of different housekeeping genes in INS-1E cells. INS-1E cells were left untreated or treated with either IL-1 (100 U/ml) or IL-1 (10 U/ml) + IFN- (100 U/ml) for 24h. GAPDH, -actin and srp-14 mRNA expression were assayed by real time PCR. Results are mean ± SEM of 4 independent experiments; * p<0.05 vs untreated cells by Student’s t test.

Supplementary Figure A2. PTPN22 is not or poorly expressed in primary FACS-purified rat -cells, human islets or INS-1E cells. PTPN22 mRNA expression was assayed in the same samples as in Fig. 1 and in rat spleen and lymph nodes, used as positive controls. Results are mean ± SEM of 3-5 independent experiments. N.S., non stimulated controls.

0.E+00

1.E+02

2.E+02

3.E+02

4.E+02

5.E+02

6.E+02

- IL IFN IL+IFNIn

s2/G

AP

DH

- siCtrl siPTPN2

0.E+00

5.E+03

1.E+04

2.E+04

2.E+04

3.E+04

3.E+04

4.E+04

- IL IFN IL+IFN

Ins1

/GA

PD

H

- siCtrl siPTPN2

**

**

**

****

**

******

0

10

20

30

40

50

- Palmitate0.5 mM

28 mMglucose

IL-1 + IFN DMSO CPA 25 µM

Apo

ptos

is (

%)

NT siCtrl si PTPN2 #1 si PTPN2 #2

a

a

a

b

a

Supplementary Figure A3. PTPN2 inhibition does not influence insulin mRNA expression in INS-1E cells. INS-1E were transfected as described in Fig. 3 and left untreated or treated with either IL-1 (100 U/ml), IFN- (100 U/ml) or IL-1 (10 U/ml) + IFN- (100 U/ml) for 24h. Insulin 1 and insulin 2 mRNA expression were assayed by RT-PCR and normalized for the housekeeping gene GAPDH. Results are mean ± SEM of 4 independent experiments; * p<0.05, ** p<0.01 and *** p<0.001 vs untreated cells by Student’s t test.

Supplementary Figure A4. PTPN2 inhibition does not exacerbate palmitate or CPA-induced cell death in INS-1E cells. INS-1E cells were left untransfected (NT), or transfected with 30 nM of either siCtrl, siPTPN2 #1 or siPTPN2 #2. After 2 days of recovery, cells were left untreated, or treated for 24h with 0.5 mM palmitate, 28 mM glucose, IL-1 (10 U/ml) + IFN- (100 U/ml) or 25 µM CPA as indicated. The control condition for CPA (DMSO) contained a similar dilution of DMSO. Apoptosis was evaluated using HO/PI staining. Results are mean ± SEM of 4 experiments; a: p<0.001 vs untreated NT or untreated transfected with the same siRNA; b: p<0.001 vs IL-1 + INF--treated NT & siCtrl; ANOVA followed by Student’s t test with Bonferroni correction.

-TUBULIN

ERK

phospho-ERK

PTPN

A

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

- 30’ 2h 4h 14h 24h

IL + IFN

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

-TUBULIN

IRphospho-IR

PTPN2

C

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

- 30’ 2h 4h 14h 24h

IL + IFN

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

-TUBULIN

EGFR

phospho-EGFR

- 1’ 5’ 15’ 30’ 1h

EGF

PTPN2

B

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

NT

siC

trl

siP

TP

N2

#1

siP

TP

N2

#2

Supplementary Figure A5. siRNA-mediated PTPN2 knockdown does not influence ERK or EGFR phophorylation, but increases IR phosphorylation. INS-1E cells were left untransfected (NT), or were transfected with 30 nM of either siCtrl, siPTPN2 #1 or siPTPN2 #2. After 2 days of recovery, cells were left untreated, or treated for different time points with IL-1 (10 U/ml) + IFN- (100 U/ml) or rrEGF (100 ng/ml) as indicated. (A) phospho-ERK and total ERK; (B) phosphor-EGFR and total EGFR; (C) phospho-IR and total IR were evaluated by Western blot. PTPN2 and -tubulin proteins were probed to ascertain accurate inhibition and equal loading respectively. Each result is representative of 4 independent experiments.

0

15

30

45

- IFN IL + IFN TNF + IFN

Apo

ptos

is (

%)

NT siCtrl siPTPN2 siPTPN2 #2 siSTAT1 siPTPN2 + siSTAT1 siPTPN2 #2 + siSTAT1

a,c,d

b ag

a

a,e,f

a

a,g,h

a,e

Supplementary Figure A6. Double knockdown of PTPN2 (by two different siRNAs) and STAT1 protects INS-1E cells from cytokine-induced apoptosis. INS-1E cells were left untransfected (NT), or were transfected with 60 nM of a control siRNA (siCtrl) or with 30 nM of either a pool of siRNAs targeting PTPN2 (siPTPN2), or another siRNA targeting PTPN2 (siPTPN2 #2), or a siRNA targeting STAT1 (siSTAT1), or double transfected with 30 nM of both siPTPN2 and siSTAT1, or siPTPN2 #2 and siSTAT1. After 2 days of recovery, cells were left untreated, or treated for 24h with IFN- (100 U/ml), IL-1 (10 U/ml) + IFN- (100 U/ml) or TNF- (1000 U/ml) + IFN- (100 U/ml). Apoptosis was then evaluated using HO/PI staining. Results are mean ± SEM of 4 independent experiments; a: p<0.001 and b: p<0.01 vs untreated NT or untreated transfected with the same siRNA; c: p<0.001 vs IFN--treated NT & siCtrl; d: p<0.001 vs IFN--treated siSTAT1, siPTPN2 + siSTAT1 & siPTPN2 #2 + siSTAT1; e: p<0.001 vs IL-1 + INF--treated NT & siCtrl; f: p<0.001 vs IL-1 + INF--treated siSTAT1, siPTPN2 + siSTAT1 & siPTPN2 #2 + siSTAT1; g: p<0.001 vs TNF- + INF--treated NT & siCtrl; h: p<0.001 vs TNF- + INF--treated siSTAT1, siPTPN2 + siSTAT1 & siPTPN2 #2 + siSTAT1; ANOVA followed by Student’s t test with Bonferroni correction.