Jan 19, 2015 - Supplementary Figure 3. 30. 60. 90. 120. 150. 0. 200. 400. 600. Days. Glucose (mg/dl). βraKO raptorf/f. *. *. * a. 0. 30. 60. 90. 120. 0. 100. 200.
Type of file: PDF Size of file: 0 KB Title of file for HTML: Supplementary Information Description: Supplementary figures and supplementary tables. Type of file: PDF Size of file: 0 KB Title of file for HTML: Peer review file Description:
Raptor
pS6 (235)
p4E-BP1 (37)
S6 KO
βra
tor f/f
rap
KO
βra
tor f/f
rap
KO
βra
tor f/f
rap
KO
βra
tor f/f
rap
KO
tor f/f
KO
tor f/f βra
rap
βra
rap
KO
tor f/f βra
rap
KO
tor f/f βra
rap
Supplementary Figure 1
Tubulin 5050 kd
pS6 (240)
4E-BP1
kDa
36 kd
36 kDa
20 kd
20kDa
150 kd kDa 150
36 kd
36 kDa
20 kd 20kDa
36 kDa
Supplementary Figure 1: Full western blots of Raptor, tubulin, pS6 (240), pS6 (235), p4E-BP1 and 4E-BP1 in raptorf/f and β raKO mice. Three membranes with samples from the same mice were used for Figure 1a. Different antibodies and time exposure were used to obtain the images in the figure. Membranes were cut around 100 kDa before probing with antibodies to increase the number of antibodies used per membrane.
1
Supplementary Figure 2 a
60 Days pS6 (240), DAPI
Insulin, pS6 (240), DAPI
βraKO
raptorf/f
Insulin, DAPI
b
30 days
Insulin Merge
Glucagon
GFP
βraKO; CAG-GFP
Supplementary Figure 2. Assessment of mTORC1 activity by S6 phosphorylation and recombination efficiency in β raKO; CAG-GFP reporter mouse. (a) Immunostaining for insulin (green), pS6 (S240) (red) and DAPI (blue) in raptorf/f and
βraKO mice at 60 days of age. Scale bars, 20 µM. Images are representative of 3 mice. (b) Immunostaining for glucagon (red), insulin (blue) and GFP (green) in pancreas from 30 day-old βraKO; CAG-GFP mouse. Scale bar 50 µM. Image is representative of 3 mice.
2
Supplementary Figure 3 a raptorf/f
βraKO
*
Glucose (mg/dl)
600
*
400
* 200 0 30
60
90
120
150
Days
c raptorf/f
Glucose (mg/dl)
400
βraptorf/+
300 200 100 0
0
30
60
Time (min)
90
120
Glucose (% of basal)
b
raptorf/f
100
βraptorf/+
80 60 40 20 0
0
15
30
Time (min)
45
60
Supplementary Figure 3. Fed glucose levels in control and βraKO females and glucose tolerance and insulin sensitivity in raptor heterozygous mice. (a) Random blood glucose levels in female raptorf/f and βraKO mice during the first 150 days of age (n=10). (b) Intraperitoneal glucose tolerance test in raptorf/f and βraptorf/+ at 60 days of age (n=5). (c) Insulin tolerance test (ITT) in raptorf/f and βraptorf/+ at 60 days of age. Data are shown as means ± s.e.m, *P < 0.05; Nonparametric U test (MannWhitney).
3
Glucose (mg/dl) 0 0
0
e
30 60
Time (min)
500
30 Days
400
raptorf/f βraKO
300
200
100
90 120
0.1
Cell size (fold change)
0.2
0.0 KO
2
0.3
βr a
0 /f
4
TUNEL positive/ insulin positive cell (%)
6
0.4
pt or f
1
c
ra
2
8
βr aK O
b
ra pt or f /f
3
Ki67 positive/ insulin positive cell (%)
4
βr aK O
ra pt or f /f
βr aK O
r f/f
to
ra p
a β-cell area/pancreas area (a.u.)
Supplementary Figure 4 Newborns
d 1.2
0.9
0.6
0.3
0.0
Supplementary Figure 4. Analysis of morphology in newborns and glucose tolerance at 30 days of age. (a) Assessment of β-cell area in control and βraKO mice during the first 24 hours of life (neonates) (n=6). (b) β-cell proliferation in sections from control and βraKO neonates stained for Ki67 and insulin (n=6). (c) TUNEL assay in sections from control and βraKO neonates stained (n=6). (d) Measure of cell size expressed as a fold change in the same group of mice (n=6). (e) Intraperitoneal glucose tolerance test in 30 day-old mice (n=5). Data expressed as means ± s.e.m., *P < 0.05. Nonparametric U test (Mann-Whitney).
4
b
c
d Insulin, Glucagon, DAPI
Insulin, Synaptophysin, DAPI
a
Insulin, Pancreatic Polypeptide, Insulin, Somatostatin, DAPI DAPI
Supplementary Figure 5 raptorf/f βraKO
Supplementary Figure 5. Assessment of different endocrine cells in sections from control and βraKO mice at 30 days of age. Immunostaining for insulin (green), (a) synaptophysin (red), (b) glucagon (red), (c) somatostatin (red) (d) and pancreatic polypeptide (red) and counterstained with DAPI (blue) in raptorf/f and βraKO mice at 30 days of age. Scale bar 50 µM. Images are representative of 3 mice per group.
5
Insulin, Glut2, DAPI
Insulin, Pax6, DAPI
Insulin, MafA, DAPI
Glucagon, PDX1, Insulin
Supplementary Figure 6
a raptorf/f
b
c
d
βraKO
Supplementary Figure 6. Markers of β-cell maturation in raptorf/f and βraKO mice at 30 days of age. Immunostaining for (a) insulin (blue), glucagon (green) and PDX1 (red). DAPI (blue), insulin (green) and (b) MafA (red), (c) Pax6 (red) and (d) Glut2 (red) in raptorf/f and βraKO mice at 30 days of age. Scale bar 18 µM (a,c,d) and 9 µM (b). Images are representative of 3 mice per group.
6
Supplementary Figure 7 90 days
βraKO
raptorf/f
β-catenin, Insulin, DAPI
Supplementary Figure 7. Staining for β-catenin to analyze cell size in raptorf/f and βraKO mice at 90 days of age. Cell size analysis in sections from 90 day-old mice stained for insulin (green), β-catenin (red) and DAPI (blue). Scale bar 50 µM. Images are representative of 5 mice per group.
7
Rictor'
Wt''''''MIP,βrakof/f'
f/f f/f' Raptor Wt''''''MIP,βrako
Supplementary Figure 8 mTOR' Raptor' a
b
Raptor' Rictor' pS6'(240)' Rictor' Raptor f/f MIP-βraKO f/f f/ff/f raptor ' Wt''''''MIP,βrako mTOR' p,4E,BP1' raptor mTOR' 150 kDa Raptor'
pS6'(240)' LC3,I/II' Rictor' pS6 (240) pS6'(240)'
36 kDa
p,4E,BP1' p4E-BP1 (37) Cl.'Casp3' mTOR' p,4E,BP1'
20 kDa
LC3,I/II' tubulin pS6'(240)' Tubulin' LC3,I/II'
50 kDa
Cre Cl.'Casp3' Cre' p,4E,BP1' Cl.'Casp3'
34 kDa
Tubulin' LC3,I/II' Tubulin' Cre' Cl.'Casp3' Cre'
4 Weeks after TMX
600
raptorf/f MIP-βraKOf/f (Tmx)
Glucose (mg/dl)
Tubulin' 500 Cre' 400
*
MIP-βraKOf/f (Corn oil)
300
*
200 100 0
c
0
30
60 Time (min)
90
120
raptorf/f MIP-βraKOf/f
raptorf/f MIP-βraKOf/f raptor
150 kDa
pULK (757)
140 kDa
tULK tubulin
LC3 I LC3 I/II LC3 II
BIMEL
23 kDa
140kDa
cl. Casp 3
19 kDa
50 kDa
tubulin
50 kDa
raptorf/f
MIP-βraKOf/f
*
3 2
* *
n.s.
* cl. Ca sp 3
EL
BI M
LC 3I I
pU LK
(75 7)
0
rap tor
Prtoein/tubulin or phospho/Total (Fold Change)
4
1
19-17 kDa
Supplementary Figure 8. Inducible Raptor deletion in β-cells results in impaired glucose tolerance and increased autophagic markers. (a) Assessment of mTORC1 targets by immunoblotting using antibodies for raptor, pS6 (S240), p4E-BP1 (T37), Cre and tubulin in islet lysates from raptorf/f and MIP-βraKOf/f mice after 4 weeks of TMX injection (TMX administered at 2 months of age) (n=4). (b) Intraperitoneal glucose tolerance test after 4 weeks post-TMX injection (TMX administered at 2 months of age). MIP-βraKOf/f mice injected with corn oil are shown in these experiments as controls (n=4). (c) Immunoblotting and quantification for raptor, pULK (S757), total ULK and tubulin (left panel), LC3-I (upper band) and LC3-II (autophagosomal marker) (lower band), BimEL, cleaved-Caspase 3 and tubulin (right panel) after 2 weeks of TMX injection of MIP-βraKOf/f mice (10 weeks of age). A representative image from four independent experiments is included and each lane shows the expression levels from one mouse. Data expressed as means ± s.e.m., n.s. not significant, *P < 0.05. Nonparametric U test (Mann-Whitney).
8
c 27 kDa
tubulin Tubulin 50kDa
125
1000
100 75
50
25
Bax Bax Bax Bax 20 kDa
20 kDa
tubulin tubulin tubulin tubulin 50kDa
50kDa
O
1200
MFI (CHOP)
150
βr aK
0 /f
to rf
aK O
/f
ra pt or f
ra p
CHOP CHOP
βr
raptorf/f βraKO
CHOP/GADPH (Fold Change)
a
/-
βrβar ca KaOKO s6 ; k; E βra if4e bp K Ei 2 -/f4 O; ra ebp pto 2 -/r f/f βra c K βaras6 O; k; KO Ei f4e bp 2 -/raβraK p ca to O s6 r f/f ; k; Ei f4e bp βra 2 -/βrKa Ei OKO f4e ; bp βra 2 -/ca KO s6 ; k; Ei f4e bp 2 -/βra Ei KO f4e ; bp 2-
βr rEai aKO pf4to ; erbf/f p2 /-
O
βra K
c r f/f
pto
ra
Supplementary Figure 9 b 800
600
400
200 0
Supplementary Figure 9. Assessment of CHOP and Bax in control and βraKO mice. (a) Assessments of CHOP levels by immunoblotting in islets and (b) by flow cytometry in
dispersed
islets
from
30
day-old
raptorf/f
and
βraKO
mice
(Median
Fluorescence Intensity, MFI). Data expressed as means ± s.e.m. Nonparametric U test (Mann-Whitney). (c) Immunoblotting for Bax and tubulin in raptorf/f, βraKO, βraKO;Eif4ebp2-/- and βraKO;caS6K;Eif4ebp2-/- mice. A representative gel is shown in the Figure and each lane shows the expression levels from one mouse (n=3).
9
βraKO + CQ
βraKO + 3MA
βraKO
raptorf/f + CQ
raptorf/f + 3MA
raptorf/f
Supplementary Figure 10 Insulin p62 Insulin, p62, DAPI
Supplemental Figure 10. p62 staining in control and βraKO mice treated with vehicle control or CQ and 3MA. Immunostaining for insulin (green), p62 (red) and DAPI (blue) in sections from 80 dayold raptorf/f and βraKO mice treated for eight weeks with 3MA (15 mg/kg in 0.9% saline), CQ (7 mg/kg in 0.9% saline) or vehicle (saline). Treatment started in 18 day-old mice. Scale bar 20 µM
10
Supplementary Figure 11
βraKO; caS6K; Eif4ebp2-/-
βraKO; cas6K
βraKO
raptorf/f
pS6 (240)
Insulin, pS6 (240), DAPI
Supplementary Figure 11. Phosphorylation of S6 in control, βraKO mice and βraKO mice with gain of S6K function. Immunostaining for pS6 (S240) (white, left panels and red, right panels), insulin (green), and DAPI (blue) in raptorf/f, βraKO, βraKO;caS6K and βraKO;caS6K;Eif4ebp2-/- mice at 60 days of age. Scale bar 50 µM. Images are representative of 3 mice.
11
βraKO; caS6K; Eif4ebp2-/-
βraKO; caS6K
βraKO; Eif4ebp2-/-
βraKO; Eif4ebp1-/-
βraKO
raptorf/f
Supplementary Figure 12 Insulin, Glucagon
Supplementary Figure 12. Panoramic pictures of pancreas from control, βraKO and βraKO mice with gain of S6K and/or 4E-BP/eIF4E function. Pancreatic sections from different experimental groups were stained for insulin (green) and glucagon (red). Exocrine pancreas can be distinguished by the red background. Scale bar 100 µM. Images are representative of 6 mice per group.
12
Supplementary Figure 13
a
raptorf/ff/f Raptor LC3 I/II Actin ac#n%
b
βraKO ββraKO raKO; ββraKO raKO; βraKO; βraKO βraKO -/-/-/- -/cas6k caS6K; cas6k; Eif4ebp2 Eif4ebp2 caS6K Eif4ebp2 Eif4ebp2 19-17 kDa 42 kDa
WT########caS6K########WT#########caS6K##########WT#########caS6K### pS6$(240)$ FLIPL$ ac,n$
36 kDa 55 kDa 42 kDa
Supplementary Figure 13. Assessment of autophagy in mice with S6K overactivation and evaluation of FLIPL in caS6K mice. (a) Immunoblotting for LC3 I/II (upper/lower band) and actin in different mouse models. A representative image from four independent experiments is included and each lane shows the expression levels from one mouse. (b) Immunoblotting for pS6 (S240), FLIPL and actin in islets from mice with overactivation of S6K (caS6k) and control (WT). A representative image from four independent experiments is included and each lane shows the expression levels from one mouse.
13
CPE
ac#n f
kDa
50 50 kd
rap + r tor f/f apa my cin caS + r 6K apa my cin
6K
f
CPE
caS
tor f/
f
-
50 kDa 42 kDa
CPE ac
