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.