glucose mm
DESCRIPTION
0. 10pM. 100pM. 1nM. 5. 6. 12. 18. 24. 30. Glucose mM. Insulin. Laminin β 1. Laminin β 1. Actin. Actin. S 1A. Dose-dependent increase in high glucose (HG)- and high insulin (HI)-induced laminin 1 synthesis. - PowerPoint PPT PresentationTRANSCRIPT
GlucosemM
Laminin β1
Actin
5 6 12 18 24 30 Insulin
Laminin β1
Actin
0 10pM 100pM 1nM
S 1A.
Dose-dependent increase in high glucose (HG)- and high insulin (HI)-induced laminin 1 synthesis.Quiescent MCT cells were incubated with or without different concentrations of HG or HI for 5 min and immunoblotting with laminin 1 antibody was performed on cell lysates. The blots were reprobed with an anti-actin antibody to assess loading. Representative blots from 2 independent experiments are shown.
S 1B.
HG, HI and HG+HI stimulate laminin 1 synthesis for up to 48 hours. MCT cells were incubated with HG, HI or HG+HI for 60 min or for up to 72 hours. Immunoblotting with laminin 1 antibody was performed on cell lysates. The blots were reprobed with an anti-actin antibody to assess loading. Representative blots from 3 experiments are shown. Composite data from 3 experiments
are shown in a graph; † p<0.01, * p<0.05 vs control by ANOVA.
0 1 2 12 24 48 72Glucose
(hr)
Laminin β1
Actin
0 1 2 12 24 48 720.00
0.25
0.50
0.75
1.00 * * *
Lam
inin
/Act
in
Time in hours
0 1 2 12 24 48 72Insulin
(hr)
Laminin β1
Actin
0 1 2 12 24 48 720.00.20.40.60.81.01.2
*
Lam
inin
/Act
in
Time in hours
0 1 2 12 24 48 72Glucose+Insulin
(hr)
Laminin β1
Actin
0 1 2 12 24 48 720.00.20.40.60.8
† †
†
Lam
inin
/Act
in
Time in hours
HG, HI and their combination (HG+HI) do not induce synthesis of type IV collagen and fibronectin following incubation for up to 60 min.Western blotting was performed on cell lysates using an anti-collagen type IV antibody and anti-fibronectin antibody. The lower panels show blots reprobed with anti-actin antibody to assess loading. Representative blots from 2 experiments are shown for type IV collagen. Histogram shows composite data from 3 experiments for fibronectin and the changes were not found to be significant.
S 1C.
Type IV Collagen
Actin
0 5 10 15 30 60Glucose
min
Type IV Collagen
Actin
0 5 10 15 30 60Glucose+Insulin
min
Type IV Collagen
Actin
0 5 10 15 30 60 Insulin
min
Glucosemin
FibronectinActin
0 5 10 15 30 60
0 5 10 15 30 600.0
0.2
0.4
0.6
Time in minutes
Fibronectin/Actin
0 5 10 15 30 600.0
0.2
0.4
0.6
Time in minutesFibronectin/Actin
Glucose+Insulinmin
Actin
Fibronectin
0 5 10 15 30 60
Actin
Insulinmin
Fibronectin
0 5 10 15 30 60
0 5 10 15 30 600.0
0.2
0.4
0.6
Time in minutes
Fibronectin/Actin
S 1D.
HG, HI and their combination (HG+HI) increase synthesis of laminin β1 as evident by 35S labelling studies.Quiescent MCT cells were pre-incubated with [35S]-methionine for 2 hours prior to incubation with HG or HI. Equal amounts of protein from each group was immunoprecipitated using anti-laminin 1 antibody. The protein coupled to protein A agarose beads were separated by boiling with sample buffer lacking bromophenol blue and centrifuged. The supernatants were spotted on 3mm filter paper for determining radioactivity. Composite
data from 3 experiments are shown in a graph; ‡ p<0.001, † p<0.01, * p<0.05 vs control by ANOVA.
†
Glucose (min)0 5 10 15 30 60
0
40
80
120
[35
S]
labe
lled
lam
inin
β1
(% o
f co
ntro
l) *
0 5 10 15 30 600
50
100
150
[35
S]
labe
lled
lam
inin
β1
(% o
f co
ntro
l) * *
Insulin (min) [35
S]
labe
lled
lam
inin
β1
(%
of
cont
rol)
0 5 10 15 30 600
40
80
120‡ ‡
Glucose+Insulin (min)
S 2A.
HG, HI and HG+HI induced laminin 1 synthesis in glomerular epithelial cells.Glomerular epithelial cells were treated with HG, HI and HG+HI for the time duration as shown in figure. Immunoblotting with laminin 1 antibody was performed on cell lysates. The lower panels in each figure show blots reprobed with anti-actin antibody to assess loading. Representative blots from 3 experiments are shown.
† p<0.01, * p<0.05 vs control by ANOVA.
0 5 10 15 30 60
Laminin β1
Glucosemin
Actin
*
0 5 10 15 30 600.0
0.2
0.4
0.6
†
La
min
in/A
ctin
Time in minutes
0 5 10 15 30 60
Laminin β1
Insulinmin
Actin
0 5 10 15 30 600.000.250.500.751.00
* *
La
min
in/A
ctin
Time in minutes
0 5 10 15 30 60
Laminin β1
Glucose+Insulinmin
Actin
0 5 10 15 30 600.00.20.40.60.81.0
* * *
La
min
in/A
ctin
Time in minutes
Laminin β1 synthesis, induced by the three conditions in glomerular epithelial cells, was inhibited by cycloheximide but not by actinomycin D.MCT cells were pre-incubated with either actinomycin D or cycloheximide prior to incubation with or without HG, HI or HG+HI. Actinomycin D did not inhibit laminin 1 synthesis but cycloheximide did in cells treated with HG,
HI and HG+HI. Loading was assessed by immunoblotting with actin antibody. ‡ p<0.001, † p<0.01, * p<0.05 by ANOVA.
S 2B.
0.0
0.2
0.4
0.6
0.8* †
La
min
in/A
ctin
Glucose+Insulin – +–
+– – +– –++
+
– –– –
– +
Laminin β1
Actin
Cycloheximide(10μm)Actinomycin(10μm)
0.000.100.200.300.400.50
* *
La
min
in/A
ctin
Glucose – +–
+– – +– –++
+
– –– –
– +
Laminin β1
Actin
Cycloheximide(10μm)Actinomycin(10μm)
0.0
0.2
0.4
0.6
0.8
†‡
La
min
in/A
ctin
Insulin – +–
+– – +– –++
+
– –– –
– +
Laminin β1
Actin
Cycloheximide(10μm)Actinomycin(10μm)
S 3.
Cycloheximide induced p38 MAPKinase phosphorylation but not HG, HI or HG+HI.Cells were pre-incubated with cycloheximide, followed by treatment with or without HG, HI or HG+HI. Cycloheximide induced p38 MAPkinase phosphorylation but not HG, HI, or both together. Representative blots from 3 independent experiments are presented.
Cycloheximide(10μm)Glucose
p38 MAPK
+ – +–+– – +
P. p38 MAPK
Insulin
p38 MAPK
P. p38 MAPK
+ – +–+– – +
Cycloheximide(10μm)
+ – +–+– – +Glucose+Insulin
p38 MAPK
P. p38 MAPK
Cycloheximide(10μm)
S 4.
LY294002 and rapamycin block HG-, HI- and HG+HI-induced laminin 1 synthesis. Quiescent MCT cells were incubated with or without 25 M LY294002, an inhibitor of PI3-kinase (A), or 22nM rapamycin, an inhibitor of mTOR (B), for 1 hour prior to treating the cells with HG, HI or HG+HI for 5 min. Immunoblotting with laminin 1 antibody was performed on cell lysates. The blots were reprobed with an anti-actin antibody assess loading. Representative blots from 3 independent experiments are shown.
A B
LY 294002(25µM)GlucoseLamininActin+–+– +––++–+– +––+InsulinLY 294002(25µM)LamininActin+–+– +––+LY 294002(25µM)LamininActinGlucose+Insulin
Rapamycin(22nM) Glucose
Laminin
Actin
+ – +–+– – +
+ – +–+– – +Insulin
Rapamycin(22nM)
Laminin
Actin
+ – +–+– – +
Rapamycin(22nM)
Laminin
Actin
Glucose+Insulin
S 5.
A B
DN-PI3-K
GlucoseVector
– +–+– – +
– –++ +
P. Akt
Insulin
DN-PI-3K Vector
– +–+– – +
– –+++
P. Akt
Glucose+Insulin
DN-PI-3K Vector
– +–+– – +
– –+++
P. Akt
DN-mTOR
GlucoseVector
– +–
+– – +– –+++
P.p70S6K
Insulin
DN-mTOR Vector
– +–
+– – +– –+++
P.p70S6K
Glucose+Insulin
DN-mTOR Vector
– +–+– – +
– –+++
P.p70S6K
Expression of dominant negative PI3-kinase and kinase-dead mTOR constructs block phosphorylation of their downstream targets. These constructs do not carry a tag. Success of mutant transfection was demonstrated functionally by showing that HG-, HI- and HG+HI-induced increment in phosphorylation of Akt and p70S6Kinase, downstream substrates for PI3-kinase and mTOR, respectively, was blocked.