Diapositive 1

1
Supplementary Table A1. List of siRNAs used in
this study
Name Distributor Sequence
       
siCtrl Allstars 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 siSTAT1 BLOCK-iT Stealth Select siRNA Invitrogen, Paisley, UK 5-CCCUAGAAGACUUACAAGAUGAAUA-3
2
Supplementary Table A2. List of primers used for
real time PCR
  Std Std   real time PCR real time PCR
  Sequence (5- 3) (bp)   Sequence (5- 3) (bp)
Rat GAPDH F ATGACTCTACCCACGGCAAG 975 F AGTTCAACGGCACAGTCAAG 118
Rat GAPDH R TGTGAGGGAGATGCTCAGTG 975 R TACTCAGCACCAGCATCACC 118
Human GAPDH F CTGAGAACGGGAAGCTTGTC 555 F CAGCCTCAAGATCATCAGCAA 106
Human GAPDH R AGGTCAGGTCCACCACTGAC 555 R TGTGGTCATGAGTCCTTCCA 106
Rat b-actin F ATGGTGGGTATGGGTCAGAA 918 F CTGTGCCCATCTATGAGGGT 133
Rat b-actin R CAGTGAGGCCAGGATAGAGC 918 R CTCTCAGCTGTGGTGGTGAA 133
Rat srp-14 F AGACCAGGATGGTGTTGCTC 436 F CGTGTTCATCACCCTGAAGA 109
Rat srp-14 R TAGACCCTTCCGTGAAAACG 436 R CGTGGCCCTTAACAGACACT 109
Mouse/rat/human PTPN2 F TGTCCGCTGTGGTAGTTCC 328 F ATCGAGCGGGAGTTCGA 110
Mouse/rat/human PTPN2 R AATGGCAGCATGTGTTCG 328 R TCTGGAAACTTGGCCACTC 110
Mouse/rat/human PTPN22 F ATTTGACATGCCCTCCCT 398 F GGCATGGACCAAAGAGAAAT 102
Mouse/rat/human PTPN22 R ATCATCCTCCAGAAGTCCAG 398 R CCTTTTCAGCTTCAGAAATTCA 102
Rat Insulin 1 F CATCAGCAAGCAGGTCATTG 316 F ACCTTTGTGGTCCTCACCTG 118
Rat Insulin 1 R TGCAGCACTGATCCACAATG 316 R AGCTCCAGTTGTGGCACTTG 118
Rat Insulin 2 F TGACCAGCTACAGTCGGAAA 390 F TGTGGTTCTCACTTGGTGGA 108
Rat Insulin 2 R GTTGCAGTAGTTCTCCAGTTGG 390 R CTCCAGTTGTGCCACTTGTG 108
Supplementary Table A3. Lysis buffers used for
Western blot
Laemmli Buffer 25 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
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Supplementary Table A4. List of antibodies used
for Western blot analysis
Antibody Company Reference Dilution
       
PTPN2 RD Systems, Abingdon, UK clone 252294 11000
STAT1 Santa Cruz Biotechnology, Santa Cruz, CA.,U.S.A. sc-346 11000
Insulin Rb Santa Cruz Biotechnology, Santa Cruz, CA.,U.S.A. sc-711 11000
phospho-STAT1 (Y701) Cell Signaling, Danvers, MA, USA 9171 11000
phospho-STAT3 (Y705) Cell Signaling, Danvers, MA, USA 9131 11000
STAT3 Cell Signaling, Danvers, MA, USA 9132 11000
phospho-p44/42 MAPK Cell Signaling, Danvers, MA, USA 4377 11000
p44/42 MAPK Cell Signaling, Danvers, MA, USA 9102 11000
phospho-EGFR Cell Signaling, Danvers, MA, USA 2236 11000
EGFR Cell Signaling, Danvers, MA, USA 2232 11000
phospho-Insulin R (pYpY1162/1163) Biosource, Camarillo, CA, USA 44-804G 11000
a-tubulin Sigma, Bornem, Belgium T9026 15000
HRP-conjugated anti-rabbit IgG Lucron Bioproducts, De Pinte, Belgium 711-036-152 15000
HRP-conjugated anti-mouse IgG Lucron Bioproducts, De Pinte, Belgium 715-036-150 15000
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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-1b (100 U/ml) or IL-1b (10
U/ml) IFN-g (100 U/ml) for 24h. GAPDH, b-actin
and srp-14 mRNA expression were assayed by real
time PCR. Results are mean SEM of 4 independent
experiments plt0.05 vs untreated cells by
Students t test.
Supplementary Figure A2. PTPN22 is not or poorly
expressed in primary FACS-purified rat b-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.
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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-1b (100 U/ml), IFN-g (100 U/ml) or IL-1b (10
U/ml) IFN-g (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 plt0.05, plt0.01 and plt0.001
vs untreated cells by Students 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-1b (10 U/ml) IFN-g (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
plt0.001 vs untreated NT or untreated transfected
with the same siRNA b plt0.001 vs IL-1b
INF-g-treated NT siCtrl ANOVA followed by
Students t test with Bonferroni correction.
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Supplementary Figure A5. siRNA-mediated PTPN2
knockdown does not influence ERK or EGFR
phophorylation, but increases IRb
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-1b (10 U/ml) IFN-g (100 U/ml) or rrEGF
(100 ng/ml) as indicated. (A) phospho-ERK and
total ERK (B) phosphor-EGFR and total EGFR (C)
phospho-IRb and total IRb were evaluated by
Western blot. PTPN2 and a-tubulin proteins were
probed to ascertain accurate inhibition and equal
loading respectively. Each result is
representative of 4 independent experiments.
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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-g (100 U/ml), IL-1b (10 U/ml) IFN-g
(100 U/ml) or TNF-a (1000 U/ml) IFN-g (100
U/ml). Apoptosis was then evaluated using HO/PI
staining. Results are mean SEM of 4 independent
experiments a plt0.001 and b plt0.01 vs
untreated NT or untreated transfected with the
same siRNA c plt0.001 vs IFN-g-treated NT
siCtrl d plt0.001 vs IFN-g-treated siSTAT1,
siPTPN2 siSTAT1 siPTPN2 2 siSTAT1 e
plt0.001 vs IL-1b INF-g-treated NT siCtrl f
plt0.001 vs IL-1b INF-g-treated siSTAT1, siPTPN2
siSTAT1 siPTPN2 2 siSTAT1 g plt0.001 vs
TNF-a INF-g-treated NT siCtrl h plt0.001 vs
TNF-a INF-g-treated siSTAT1, siPTPN2 siSTAT1
siPTPN2 2 siSTAT1 ANOVA followed by
Students t test with Bonferroni correction.