supplementary table a1. list of sirnas used in this study
DESCRIPTION
Supplementary Table A1. List of siRNAs used in this study. Supplementary Table A2. List of primers used for real time PCR. Supplementary Table A3. Lysis buffers used for Western blot. Supplementary Table A4. List of antibodies used for Western blot analysis. *. GAPDH. b -actin. srp-14. - PowerPoint PPT PresentationTRANSCRIPT
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.