Ruolo del Metilgliossalenello sviluppo delle … Nigro Cecilia...Ruolo del Metilgliossalenello...
Transcript of Ruolo del Metilgliossalenello sviluppo delle … Nigro Cecilia...Ruolo del Metilgliossalenello...
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Ruolo del Metilgliossale nello sviluppo delle
complicanze vascolari associate al diabete mellito
Cecilia Nigro
URT-Genomica del Diabete IEOS/CNRDiSMeT, Università di Napoli “Federico II”
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Glucose
Methylglyoxal
Hypoxia
Sources and Destiny of Methylglyoxal
AGEs
Glyoxalase 1
Glyoxalase 2
GSHS-D-LactoylGSH
D-Lactate
>99%ThreonineAcetone
Lipid
Glycatedptoteins
extracellularAGEs
RAGE
Oxidative stress
Pro-inflammatory state
+ NFkB
- Nrf2
GSHdepletion
Ageing
DicarbonylStress=
Pyruvate
L-Lactate
Glucose
Fructose6-diphosphate
G3P DHAP
0.1%
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MGO
Methylglyoxal & Diabetes
[McLellan et al. Clinical Science 1994]
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MGO
[Fleming T et al. Diabetologia 2012]
Even if normalized on glucose levels,intracellular MG is higer in diabetic patients
[Maessen DE et al. Diabetes Care 2015]
Post-prandial hyperglycaemia induces aglucose-similar trend of MGO increase
Methylglyoxal & Diabetes
HbA1c 7
HbA1c 8 to 7HbA1c 8
Healthy
[M. Lind et al. Diabetologia 2010]
HbA1c can only partly explain the latediabetic complications
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Methylglyoxal & Complications
Plasma MGO and MG-H1 are risk predictors for progression of diabetic kidney disease. [Jensen et al., Diabet Med 2016; Beisswenger et al., Diabetes Care 2013]
MG-H1 is independently associated with microvascular complication progression.[Genuth e al., Diabetes 2015]
Decreased Glo1 expression is a driver of coronary heart disease.[Makinen et al., PLOS genetics 2014]
Dicarbonyl stress in plasma likely contributes to CVD risk through induction of dyslipidaemia.[Rabbani N, et al. Diabetes 2011]
Inducer of Glo1 expression corrects insulin resistance and improves vascular function in overweight and obese subjects.[Xue et al., Diabetes 2016]
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Methylglyoxal in Experimental Models
Glo1 overexpression attenuates the life-shortening effect of glucose by preventingMG-modification of mitochondrial proteins in C. Elegans, and prevents the age-related endothelial dysfunction in rats.[Morcos et al., Aging Cell 2008; Jo-Watanabe et al., Aging Cell 2014]
MGO alters blood vessel formation in Zebrafish and via a RAGE-dependentmechanism in mice.[Jorgens et al., Diabetes 2015; Liu et al., PLoS ONE 2012]
Regulation of Glo1 plays a role in the prevention of early renal impairment inexperimental diabetes, but also independently of hyperglycaemia in ApoE-/- mice.[Geoffrion et al., Physiol Rep 2014]
Hyperglycaemia-induced impairment of endothelial-dependent vasorelaxation ismediated by increased intracellular MGO levels in a pathway dependent on oxidativestress.[Brouwers et al., Diabetologia 2010]
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IRS-1
PI3K
Akt
eNOS
KRAS
ERK 1/2
ECE-1
MGO
nucleus
miR-190a
ET-1
NO
endothelial cell
IR
P
KRAS mRNAAAA
Impaired vasodilation
X
Methylglyoxal impairs endothelial insulin-sensitivity
MGO exogenous exposure
Glo1 inhibitor
Endothelialinsulin-resistance
[Nigro C. et al., Diabetologia 2014;Nigro C. et al., Biochim Biophys Acta 2017]
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AIM
To investigate the effect of MGO on glucose homeostasis
and its role on the development of
vascular complications associated with diabetes
Glo1KD MAECsGlo1KD mice
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[Bierhaus A et al. Cell Metabolism 2012]
Glo1KD mice show thermal and mechanical hyperalgesia, a sign ofDiabetic Neuropathy.
[Giacco F et al. Diabetes 2014]
Glo1KD mice show alterations of kidney morphology (↑ mesangialfraction volume and GBM tickness), mimicking Diabetic Nephropathy.
Glo1KD mice
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Glo1KD mice
Characterization of Glo1KD mice
Glo1KD mice show a decreased tissue expression of glo1 and a 50% increase of serum MGO levels
0
500
1000
1500
2000
2500
WT Glo-/+
Seru
mM
GO
(nM
)
***
Glo1KD
******
***
0,0
0,2
0,4
0,6
0,8
1,0
1,2
Glo
1 ex
pres
sion
(nor
mal
ized
ratio
)
WT
Glo-/+
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Glo1KDWT
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Glo1KD mice
5 months 10 months
Variable wt mice Glo1kd mice Test T wt mice Glo1kd mice Test T
Body weight (g) 25.4±0.8 24.5±0.7 n.s. 32.6±1.5 35.1±1.8 n.s.
Fasting glycaemia(mg/dl) 105.9±5.3 97.1±3.5 n.s. 114.6±8.4 106.4±6.0 n.s
Fed glycaemia(mg/dl) 138.9±4.1 145.6±6.9 n.s. 143.6±3.1 137.3±4.4 n.s.
GTT (AUC) 8721±960 9921±849 n.s 12990±1595 23944±2300 p
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Glo1KD mice
050
100150200250300350400450500
-15 0 15 30 45 60 75 90 105 120 135
Bloo
dgl
ucos
ele
vels
(mg/
dl)
Time (min)
**** ****
***
0
5000
10000
15000
20000
25000
30000
GTT
AU
C (m
g/dl
·min
)
***
10 months of age
wt mice Glo1KD mice
0
50
100
150
200
250
300
350
-15 0 15 30 45 60 75 90 105 120 135Blo
odgl
ucos
ele
vels
(mg/
dl)
Time (min)
0
5000
10000
15000
20000
25000
30000
GTT
AU
C (m
g/dl
·min
)
n.s.
5 months of age
wt mice Glo1KD mice
Metabolic characterization of Glo1KD mice
Glo1KD mice develop an age-dependent glucose intolerance
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Glo1KD mice
5 months 10 months
Variable wt mice Glo1kd mice Test T wt mice Glo1kd mice Test T
Body weight (g) 25.4±0.8 24.5±0.7 n.s. 32.6±1.5 35.1±1.8 n.s.
Fasting glycaemia(mg/dl) 105.9±5.3 97.1±3.5 n.s. 114.6±8.4 106.4±6.0 n.s
Fed glycaemia(mg/dl) 138.9±4.1 145.6±6.9 n.s. 143.6±3.1 137.3±4.4 n.s.
GTT (AUC) 8721±960 9921±849 n.s 12990±1595 23944±2300 p
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Glo1KD mice
(*: KD mice vs WT mice; #: 3 and 30 minutes vs time 0)
0
1
2
3
4
5
6
0 5 10 15 20 25 30
Insu
linem
ia(n
g/m
l)
Time (min)
***
##
##
##
0
10
20
30
40
50
60
70
80
90
Insu
lin se
cret
ion
(AU
C)
*
WT mice Glo1KD mice
Metabolic characterization of Glo1KD mice
Glo1kd mice show an alteration of glucose-stimulated insulin secretion at 10 months of age
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Metabolic characterization of Glo1KD miceGlo1KD mice
0,00
0,50
1,00
1,50
2,00
2,50
3,00
TNF alpha
0,00
0,50
1,00
1,50
2,00
2,50
3,00
MCP1
**
0,00
0,50
1,00
1,50
2,00
2,50
3,00
IL-1beta
**
mRN
Ale
vels
(REU
)
WTmice
Glo1KDmice
WTmice
Glo1KDmice
WTmice
Glo1KDmice
WT Glo1KD
F4/80
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Glo1KD mice
5 months 10 months 18 months
Variable wt mice Glo1kd mice
wt mice Glo1kd mice wtmice
Glo1kd mice
Systolic blood pressure(mmHg)
124±3 147±4* 115±3 142±4* 144±3 159±4*
Vascular function of Glo1KD mice
Glo1KD mice show a higher systolic blood pressure,accompanied by an impaired endothelial-dependent vasodilation
and the development of left ventricular hypertrophy
Relative wall thickness (RWT)
Concentric hypertrophy
Eccentric hypertrophy
Vasodilation(% of residual vasoconstriction)
40±0.2 65±0.7* 23±0.2 43±0.3* n.s. n.s.
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Summary 1Glo1KD mice
Next question: Is there a cause-effect relationship between the metabolic and vascular phenotype?
Impairment of GLUCOSE
HOMEOSTASIS
Impairment ofVASCULAR FUNCTION
Glo1KD mice show an age-dependent impairment of glucose tolerance
Glucose intolerance is likely linked to the impairment of insulin secretion
and pancreatic dysfunction, rather than peripheral insulin-resistance
Glo1KD mice show an impaired endothelial-dependent vasodilation and
increased systolic blood pressure
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WT mice
Glo1KDmice
Effect of MGO accumulation on angiogenesis
Endothelial outgrowth from the aortic rings of Glo1KD mice is highly impaired compared to those from WT mice
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***
0,0
0,2
0,4
0,6
0,8
1,0
1,2
WT MAECs
Glo1KDMAECs
0,0
0,5
1,0
1,5
2,0
2,5
3,0
3,5
WT MAECs
Glo1KDMAECs
Intr
acel
lula
r M
ethy
lgly
oxal
(nm
ol m
g-1)
0.0
0.2
0.4
0.6
0.8
1.0
1.2
Glo
1 m
RN
Ale
vels
(RE
U)
0.0
0.5
1.0
1.5
2.0
2.5
3.5
3.0
***
Glo1KD MAECs
Characterization of Glo1KD MAECs
Glo1KD MAECs show a 50% decrease of Glo1 expression and a 5-fold increase of intracellular MGO accumulation
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0
1
2
3
4
5
6
7
8
9
10
11
0 12 24 36 48 60 72 84 96
Prol
ifera
tion
(fol
d ov
er ti
me
0)
****
**
WT MAECsGlo1KD MAECs
Time (h)
WT MAECs
Glo1KD MAECs
- FBS + FBS
Mig
ratio
n(f
old
incr
ease
ove
r ba
sal)
***
WT MAECs
Glo1KD MAECs
0
1
2
3
4
5
6
7
8
9
10
WT MAECs
Glo1KD MAECs
- FBS + FBS
Inva
sion
(fol
d in
crea
se o
ver
basa
l)
*
0
1
2
3
4
5
6
7
8
9
10
WT MAECs
Glo1KD MAECs
Glo1KD MAECs
Effect of MGO accumulation on angiogenesis in MAECs
Glo1KD MAECs show an impaired
proliferation, migration and
invasion ability,
compared to WT MAECs
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Glo1KD MAECs
Anti-angiogenic genes mRNA expression (REU) p value
HoxA5 1,87±0,37 0.00000003
HoxD10 1,20±0,92 n.s.
Pro-angiogenic genes mRNA expression (REU) p value
HoxA3 3,37±2,29 n.s.
HoxA9 2,19±1,4 n.s.
HoxB3 1,04±0,67 n.s.
HoxA5 involvement in MGO-mediated impairment of angiogenesis
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Hox
A5
mR
NA
leve
ls (R
EU
)***
0
10
20
30
40
50
60
70
80
90
100
Hox
A5/α-
Tub
ulin
(O.D
.)
*
HoxA5
α-Tubulin
2.5
2.0
1.5
1.0
0.5
0.0
Glo1KD MAECs
HoxA5 involvement in MGO-mediated impairment of angiogenesis
Both mRNA and protein levels of the anti-angiogenic HoxA5are 2-fold increased in Glo1KD MAECs
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Grafico1
11.873749602800.3702646218
wt
kd2
Foglio1
Pro-angiogeniciAnti-angiogenici
HoxB3 variante 1 e 2wtkd2HoxA5wtkd2
15.3.1710.3715.11.1612.10
11.3.1610.55292813.12.1611.51
14.7.1610.37453315.3.1712.01
15.3.1711.754.12.1512.03
11.3.1611.52526137242.38
14.7.1611.662970605620.710.8433749242
mean11.03928216329.711.50
dev.st00.671811483224.10.1611.60
SEM0.274265889530.11.1611.148698355
test.t0.888956201622.12.1611.26
27.1.1713.10
HoxD3 newwtkd26.2.1711.91
6.11.14121.6118.1.1613.40
17.10.1413.93esp silenz11.59
cp 3.2.16118.6311.82
cp 14.10.16113.7112.52
12.14
mean117.983333333315.53
dev.st03.98950289314.00
SEM02.3033405692mean11.874
test.t0.0018031364dev.st00.3702646218
SEM0.1026929292
test.t0.00000003B3D3A3A9A5D10
HoxA3wtkd2
17.316.727171322
22.3.1712.0373116199
116.5642387815HoxD10wtkd2
12.96561914417.310.632878297
22.3.1711.922966105
mean17.073585216810.1963727522
devst06.644093734812.0443846535
SEM3.3220468674mean11.1991504519
test.t0.1172695611devst00.9245822883
SEM0.4622911442
test.t0.6817
HoxA9 variante 1 e 2wtkd2
15.3.1711.7565193816
11.3.1613.8320602189
14.7.161
15.3.1710.9979499577
11.3.16
14.7.16
mean12.1955098527
dev.st01.4671672428
SEM0.8470694026
test.t0.1524326551
HoxD3 oldwtkd2
17.6.141
17.10.1410.51
21.10.1410.78
HoxB3 variante1wtkd2
15.3.1710.37
11.3.1610.552928
14.7.1610.374533
mean10.432487
dev.st00.1043295878
SEM0.0602347156
test.t0.0007074623
HoxB3 variante2wtkd2
15.3.1711.75
11.3.1611.5252613724
14.7.1611.6629706056
mean11.646077326
dev.st00.1133176944
SEM0.0654240013
test.t0.0005899848
HoxA9 variante1wtkd2
15.3.1711.7565193816
11.3.1613.8320602189
14.7.161
mean12.7942898003
dev.st01.4676290007
SEM1.0377704186
test.t
HoxA9 variante2wtkd2
15.3.1710.9979499577
11.3.16
14.7.16
mean10.9979499577
Foglio1
0.6718114832
3.989502893
6.6440937348
1.4671672428
0.3702646218
0.9245822883
kd2
00.3702646218
wt
kd2
-
**
*
***
**
0,0
0,5
1,0
1,5
2,0
2,5**
2.5
2.0
1.5
1.0
0.5
0.0
Hox
A5
mR
NA
exp
ress
ion
(RE
U)
Scramble 5nM - + -HoxA5 siRNA 5nM -- +
n.s.
Inva
sion
(fol
d in
crea
se o
ver
basa
l) *
+ + -
-- +
WT MAECs
Glo1KDMAECs
0
1
2
3
4
5
6
7
8
9
10
11
*
Mig
ratio
n (f
old
incr
ease
ove
r ba
sal)
*
+ + -
-- +
WT MAECs
Glo1KDMAECs
0
1
2
3
4
5
6
7
8
9
10
11
**
Glo1KD MAECs
HoxA5 involvement in MGO-mediated impairment of angiogenesis
HoxA5 silencing is able to improve both migration and invasion ability of Glo1KD MAECs
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-
WTMAECs
Glo1KDMAECs
NF-κB
-p65
mR
NA
leve
ls (R
EU
)
0,0
0,5
1,0
1,5
2,0
2,5
3,0***
0.0
0.5
1.0
1.5
2.0
2.5
3.0NF-κB-p65
GAPDH
cytosol
NF-κB
-p65
/GA
PDH
(O.D
.)
*
NF-κB-p65
Cyclin G1
nucleus
NF-κB
-p65
/Cyc
linG
1 (O
.D.)
*
Glo1KD MAECs
NF-κB-p65 role in the regulation of MGO-mediatedHoxA5 overexpression
Both mRNA and protein levels of NF-kB-p65are 2-fold increased in Glo1KD MAECs
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Grafico1
36.277935129462.68601506998.126591533123.7439387463
WT
KD2
p-Akt
Densitometria lastra del 3-7-15 cp 1-7-15nota: bande bianche
p-AktmeanODtubulinameanODpAkt/tubwt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50
wt -ins154,7141,986wt -ins219,80756,3653.5234631427/3/153.5273.5941.2517.99144.3816.62nota: stim vegf 150
wt +ins185,22332,495wt +ins207,60044,15873.588024826/26/1523.4952.1931.4818.7156.848.39
wt +VEGF150172,02519,297wt +VEGF150210,21946,77741.25317998166/26/1515.5362.3931.6028.1690.2522.60
kd -ins162,5769,848kd -ins218,18754,74517.98885742995/7/1514.3865.9413.8914.2591.6424.78
kd +ins210,24157,513kd +ins203,27639,834144.3816839886
kd +VEGF150160,5617,833kd +VEGF150210,57147,12916.6203399181mean14.1862.7234.7821.6297.1615.87
background152,728163,442dv.st10.0510.705.615.6744.187.13
0.020.17-insVEGF 50ng/ml
lastre del 26-6-15 cp 18-6 p100
p-AktmeanODTubulinameanODpAkt/tub
wt -ins181,77516,039wt -ins237,56368,28023.4900410076
wt +ins199,80134,065wt +ins234,55165,26852.1924986211
wt +VEGF50185,31019,574wt +VEGF50231,46162,17831.4805879893
kd -ins178,52012,784kd -ins237,62468,34118.7061939392
kd +ins203,90338,167kd +ins236,43367,15056.8384214445
kd +VEGF50171,3775,641kd +VEGF50236,48467,2018.3942203241
background165,736background169,283
lastre del 26-6-15 cp 24-6 p60
p-AktmeanODTubulinameanODpAkt/tub
wt -ins176,62010,706wt -ins229,65968,92315.5332762648
wt +ins208,24842,334wt +ins228,59467,85862.3861593327
wt +VEGF50186,23520,321wt +VEGF50225,04664,31031.5985072306
kd -ins182,98117,067kd -ins221,35460,61828.1550034643
kd +ins219,01653,102kd +ins219,57658,84090.2481305235
kd +VEGF50180,01914,105kd +VEGF50223,15962,42322.5958380725
background165,914background160,736
lastre del 13-5-15 cp 8-5
p-AktmeanODMANCA NORMALIZZATORE
wt -ins97,1497,367
wt +ins131,32041,538
wt +VEGF50100,46610,684
wt +VEGF15097,0397,257
kd -ins95,7095,927
kd +ins141,01951,237
kd +VEGF5094,4204,638
kd +VEGF15094,0214,239
background89,782
lastre del 7-5-15 cp 30-4 P100
p-AktmeanODTubulinameanODpAkt/tub
wt -ins73,05011,771wt -ins146,94781,86614.38
wt +ins134,51673,237wt +ins176,153111,07265.94
wt +VEGF5078,20716,928wt +VEGF50186,950121,86913.89
kd -ins78,14816,869kd -ins183,419118,33814.25
kd +ins162,430101,151kd +ins175,455110,37491.64
kd +VEGF5073,48412,205kd +VEGF50114,32749,24624.78
background61,279background65,081
lastre del 25-9-15
IKBαmeanODTubulinameanODIKBα/tubulinameandev.sttest.t
wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622
wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30
wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39
kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474
kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33
kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82
background146,742background160,713
p-Akt
10.05161635495.6736980636
10.701721438144.1785639731
5.60847818227.1304694614
wt
kd
signaling pAKT
10.05161635495.6736980636
5.60847818227.1304694614
wt
kd
pAkt/tubulina (OD)
signaling eNOS
22.683367862232.4451951155
wt
kd
IKBa-NFkB
Densitometria 28.10.15 cp wt p25/kd2 p22-insVEGF 50ng/ml
p-AktmeanOD14.3.3meanODpAkt/14.3.3
wt p25 -176,35833,572wt p25 -191,06834,05798.5759168453wt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50
wt p25 +VEGF50181,72538,939wt p25 +VEGF50189,30232,291120.58777987677/3/156.7076.9412.927.8760.384.74nota: stim vegf 1507/3/151001148.8988903168117.5298069394901.66532258917/3/15100192.9469563869117.529806939470.7924584787
kd2 p22 -151,9759,189kd2 p22 -193,27336,26225.34057691256/26/1530.1874.3140.4722.3982.248.356/26/15100246.208369934174.1862488553272.45712563726/26/15100134.071546402974.186248855327.6542550773
kd2 p22 +VEGF50154,26611,480kd2 p22 +VEGF50193,07436,06331.83318082246/26/1517.7468.4437.5834.65104.9826.826/26/15100385.8082919831195.3163440198591.7988036876/26/15100211.8245924718195.3163440198151.1999742335
background142,786background157,0115/7/1516.3674.0114.1814.6197.3224.285/7/15100452.295768442489.2992749743594.777361807810/28/15100122.329858788925.706661143532.2930608623
14.3.310/28/1598.58120.5925.3431.8312/15/15100140.721001484724.666707594812.0123648366
Densitometria lastra del 3-7-15 cp 1-7-15nota: bande nere12/15/1589.65126.1622.1110.771/13/16100593.1357814234765.8291967053736.621379007
p-AktmeanODtubulinameanODpAkt/tub96.68100.1121.808.581/18/16100186.7782247738197.0159635832185.7824134586
wt -ins157,1243,440wt -ins212,98851,3686.69677620311/13/1620.74122.99158.80152.75-
wt +ins184,17530,491wt +ins201,25039,63076.939187484275.4868.69
wt +VEGF150159,6125,928wt +VEGF150207,49845,87812.9212258599138.78108.90mean100.00558.30119.08590.17mean100.00225.97200.04173.77
kd -ins157,2443,560kd -ins206,85145,2317.87070814261/18/1616.3530.5432.2230.38dv.st0.00402.9953.90256.90dv.st0.00165.37259.47256.77
kd +ins187,30833,624kd +ins217,30555,68560.38250875460.030.0060.030.426
kd +VEGF150155,7392,055kd +VEGF150204,96743,3474.740812513mean58.2072.2573.1823.4894.8518.440.2530.164
background153,684161,620dv.st40.683.3148.166.5211.5710.41
0.290.17
lastre del 26-6-15 cp 18-6 p100nota: in rosso esclusi esperimenti wt no risp a VEGF
p-AktmeanODTubulinameanODpAkt/tub
wt -ins185,66718,099wt -ins226,02759,96430.1831098659
wt +ins209,45141,883wt +ins222,42356,36074.3133427963
wt +VEGF50189,38921,821wt +VEGF50219,98653,92340.4669621497
kd -ins180,54412,976kd -ins224,01357,95022.3917169974
kd +ins214,40846,840kd +ins223,02156,95882.2360335686
kd +VEGF50172,2804,712kd +VEGF50222,51556,4528.3469141926
background167,568background166,063
lastre del 26-6-15 cp 24-6 p60
p-AktmeanODTubulinameanODpAkt/tub
wt -ins176,0819,811wt -ins222,00055,30617.7394857701
wt +ins203,86537,595wt +ins221,62554,93168.4404070561
wt +VEGF50185,34219,072wt +VEGF50217,44950,75537.5765934391
kd -ins182,55616,286kd -ins213,69847,00434.648115054wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50
kd +ins213,68247,412kd +ins211,85645,162104.98206456767/3/156.7076.947.8760.387/3/156.7012.927.874.74
kd +VEGF50179,40813,138kd +VEGF50215,67648,98226.82209791356/26/1530.1874.3122.3982.246/26/156.048.094.481.67
background166,270background166,6946/26/1517.7468.4434.65104.986/26/157.1015.0313.8610.73
5/7/1516.3674.0114.6197.3210/28/156.167.541.581.99
lastre del 13-5-15 cp 8-512/15/155.988.411.470.72
p-AktmeanOD1/13/166.9141.0052.9350.92
wt -ins97,4765,6901/18/165.4510.1810.7410.13
wt +ins140,53548,749
wt +VEGF50101,7709,984
wt +VEGF15097,8466,060mean17.7573.4319.8886.23mean6.3314.7413.2811.56
kd -ins96,2714,485dv.st9.643.5711.4919.65dv.st0.5911.9018.0817.82
kd +ins152,95761,1710.000.0010.040.430
kd +VEGF5094,9893,2030.3930.165
kd +VEGF15094,2292,443
background91,786
lastre del 7-5-15 cp 30-4 P100
p-AktmeanODTubulinameanODpAkt/tub
wt -ins77,72512,031wt -ins137,56873,52516.36
wt +ins140,32474,630wt +ins164,881100,83874.01
wt +VEGF5081,29715,603wt +VEGF50174,046110,00314.18
kd -ins81,30915,615kd -ins170,906106,86314.61
kd +ins162,26496,570kd +ins163,26899,22597.32
kd +VEGF5076,37010,676kd +VEGF50108,00743,96424.28
background65,694background64,043
lastre del 15.12.15 cp del 3.12.15 (Potito) wtp19/kd2p17
p-AktmeanODActinameanODpAkt/actin
wt -1113,69431,230wt -1150,46634,83589.65
wt -2116,89134,427wt -2151,23935,60896.68
wt +VEGF50 1126,88344,419wt +VEGF50 1150,84035,209126.16
wt +VEGF50 2121,28538,821wt +VEGF50 2154,41138,780100.11
kd2 -192,77210,308kd2 -1162,24446,61322.11
kd2 -296,53114,067kd2 -2180,15464,52321.80
kd2 +VEGF50 188,0715,607kd2 +VEGF50 1167,69652,06510.77
kd2 +VEGF50 284,9332,469kd2 +VEGF50 2144,41728,7868.58
background82,464background115,631
TubulinameanODpAkt/tub
wt -1200,53970,57444.25
wt -2215,51085,54540.24
wt +VEGF50 1216,74786,78251.18
wt +VEGF50 2218,93188,96643.64
kd2 -1213,24283,27712.38
kd2 -2213,29483,32916.88
kd2 +VEGF50 1215,84185,8766.53
kd2 +VEGF50 2208,25178,2863.15
background129,965
lasta del 13-1-16 cp del 17.12.15
p-AktmeanOD14.3.3meanODpAkt/14.3.3
wt -1102,57910,637wt -1149,25551,29720.74
wt +VEGF50 1157,64565,703wt +VEGF50 1151,37853,420122.99
wt -2146,85754,915wt -2170,71672,75875.48
wt +VEGF50 2127,43335,491wt +VEGF50 2149,62951,67168.69
kd2 -1167,57275,630kd2 -1145,58347,625158.80
kd2 +VEGF50 3187,56995,627kd2 +VEGF50 3160,56362,605152.75
wt -3168,06476,122wt -3152,80854,850138.78
wt +VEGF50 3147,39555,453wt +VEGF50 3148,87950,921108.90
background91,942background97,958
lastra del 20.1.16 wb 18.1.16 cp vedi quaderno
p-AktmeanODTubulinameanODpAkt/tubulina
wt p20 -2182,78811,443wt p20 -2207,50869,97416.35
wt p20 +VEGF50 2192,43021,085wt p20 +VEGF50 2206,56569,03130.54
kd2 p18 -1192,30620,961kd2 p18 -1202,59365,05932.22
kd2 p18 +VEGF50 1190,17618,831kd2 p18 +VEGF50 1199,51661,98230.38
background171,345background137,534
IKBa-NFkB
40.67599330046.5221844176
3.306600167811.5735223607
48.161030696710.4093376061
wt
kd
pAKT/Tub
NF-kB n-c
053.9033817395
402.9882219996256.9012048943
wt
kd2
HoxA5
0259.468648097
165.3739785067256.7718898938
wt
kd2
PARP
Densitometria 28.10.15 cp wt p25/kd2 p22
eNOSmeanOD14.3.3meanODeNOS/14.3.3
wt p25 -172,37030,350wt p25 -191,06834,05789.1153066917
wt p25 +VEGF50176,62634,606wt p25 +VEGF50189,30232,291107.1691802669
kd2 p22 -162,22020,200kd2 p22 -193,27336,26255.7056974243
kd2 p22 +VEGF50161,48219,462kd2 p22 +VEGF50193,07436,06353.9666694396
background142,020background157,011
VEGFR2
Densitometria lastra del 21-10-15 cp del 19-10-15nota: bande nere
lastre del 25-9-15
IKBαmeanODTubulinameanODIKBα/tubulinameandev.sttest.t
wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622
wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30
wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39
kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474
kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33
kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82
background146,742background160,713
lastra del 25-11-15lastra del 21-10
NF-kB p65meanOD14.3.3meanODNF-kB p65/14.3.3
wt -114,57936,699wt -144,06662,96558.28
wt +v50151,12973,249wt +v50138,67757,576127.22
kd -147,83569,955kd -158,98677,88589.82
kd +v50180,424102,544kd +v50179,32398,222104.40
kd2 -144,75166,871kd2 -174,70193,60071.44
kd2 +v50171,70393,823kd2 +v50151,83770,736132.64
background77,880background81,101
-VEGF50
lastra del 21.10
IKB-αmeanODIKB-α/14.3.3
wt -138,70058,30592.60
wt +v50146,43066,035114.69
kd -162,14481,749104.96
kd +v50172,73892,34394.01
kd2 -167,39987,00492.95
kd2 +v50147,05666,66194.24
background80,395
VEGFR2
90.193494332384.866712302922.683367862232.4451951155
wt
kd
wt
kd
kd2
NF-kB p65
wt
kd
kd2
IKB-α
Densitometria lastra del 20-11-15 cp estrazione nucleo citosol: wtp17/kd2p15 (1x106 cell)+ wtp18/kdp18 (5x105 cell)nota: bande nerecitoplnuclSUMM31.748144877837.0358623023
NF-KbmeanODGAPDHmeanODNF-kB/GAPDHActinameanODNF-kB/ActinacitoplasmaNF-kB/GAPDH
wt p17 citopl130,44366,272wt p17 citopl191,99949,096134.98wt p17 citopl90,62918,472358.77citoplasmaNF-kB/GAPDH
kd2 p15 citopl171,805107,634kd2 p15 citopl196,12353,220202.24kd2 p15 citopl119,69647,539226.41WTKD2fold
wt p18 citopl168,943104,772wt p18 citopl182,82200.00wt p18 citopl118,86946,712224.29esp 20.11134.98202.241.4982960439WTKD2
kd p18 citopl169,506105,335kd p18 citopl175,08800.00kd p18 citopl132,44660,289174.72esp 13.1.1649.0880.761.6454767726esp 20.1130.993824443588.0407348343
background64,171background142,903background72,157esp.5.2.1659.48103.681.7432309455esp 16.1231.748144877837.0358623023
esp.10.2.16209.42269.311.2859996977esp 13.1.1649.0880.76
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1esp 16.12222.2370141448259.2510361161.1665520126esp.5.2.1639.650459142769.1199073797
wt p17 nucleo87,45223,281wt p17 nucleo134,51656,72141.04mean81.1785629047128.8932870232esp.10.2.1629.917247183138.4735708332
kd2 p15 nucleo101,94337,772kd2 p15 nucleo121,29343,49886.84dev st46.882444060764.5456058631
wt p18 nucleo75,05310,882altra lastrawt p18 nucleo129,04051,24521.241.5877749299mean36.277935129462.6860150699
kd p18 nucleo74,73210,561kd p18 nucleo126,27448,47921.780.1793685306dev st8.126591533123.7439387463
background64,171background77,7951.7279377905
0.0232329513
nucleoNF-kB/CyclinG1
nucleoNF-kB/CyclinG1
WTKD2fold
esp 20.1141.0486.842.1159844055WTKD2
esp 16.1279.8485.241.0675814633esp 20.1127.3657.8933333333
esp 13.1.16151.0826939471247.08973322551.6354602024esp 16.1279.8485.24
esp 5.2.1628.9738430584115.53557862753.9875821235lamin Besp 13.1.1675.5413469736123.5448666128
esp.10.2.16esp 5.2.1628.9738430584115.5355786275lamin B
esp.10.2.16
mean75.2353790321133.6765828756
dev st55.025803797376.8793600128mean52.930042288795.5536995557
1.7767782205dev st28.655490855830.0412797958
0.13127368911.8052828871
0.0429221995
Densitometria lastra del 16-12-15 cp estrazione nucleo citosol: wtp20/kd2 p19 25.11 /kd2 p20 1.12nota: kd2 errore CERII
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p20 citopl172,08893,484wt p20 citopl190,74242,065222.24
kd2 p19 citopl140,63962,035kd2 p19 citopl158,6029,925625.04
kd2 p20 citopl218,724140,120kd2 p20 citopl202,72554,048259.25
background78,604background148,677
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1
wt p20 nucleo112,69634,612wt p20 nucleo144,31643,34979.84
kd2 p19 nucleo115,76937,685kd2 p19 nucleo147,31946,35281.30
kd2 p20 nucleo137,90759,823kd2 p20 nucleo171,14870,18185.24
background78,084background100,967
Densitometria lastra del 13.1.16 cp estrazione nucleo citosol del 11.1.16 wtp19/kd2p18
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p19 citopl127,15329,146wt p19 citopl145,80559,37949.08
kd2 p18 citopl145,84247,835kd2 p18 citopl145,65559,22980.76
background98,007background86,426
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1
wt p19 nucleo115,72917,722wt p19 nucleo162,55511,730151.08
kd2 p18 nucleo134,68536,678kd2 p18 nucleo165,66914,844247.09
background98,007background150,825
Densitometria lastra del 5.2.16 cp estrazione nucleo citosol del 1.2.16 wtp28/kd2p26
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p28 citopl185,19146,849wt p28 citopl150,35278,77059.48
kd2 p26 citopl207,59669,254kd2 p26 citopl138,37866,796103.68
background138,342background71,582
NF-KbmeanODLamin BmeanODNF-kB/Lamin B
wt p28 nucleo151,30212,960wt p28 nucleo187,60144,73028.97
kd2 p26 nucleo174,84236,500kd2 p26 nucleo174,46331,592115.54
background138,342background142,871
Densitometria lastra del 10.2.16 cp 8.2.16 estrazione nucleo citosol del wtp19/kd2p20
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p19 citopl197,50344,793wt p19 citopl193,18421,389209.42
kd2 p20 citopl223,79371,083kd2 p20 citopl198,18926,394269.31
background152,710background171,795
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1
wt p19 nucleo168,89216,182wt p19 nucleo
kd2 p20 nucleo173,43320,723kd2 p20 nucleo
background152,710background
manca normalizzatore nucleo: Lamin B (lastra 17.2.16) troppo vicina a NFKB
wt p17
kd2 p15
NF-kB/Actin
wt p18
kd p18
wt p17
kd2 p15
NF-kB/Cyclin G1
wt p18
kd p18
wt
kd2
NF-kB/cyclG1
nucleo
wt
kd2
NF-kB/GAPDH
Citoplasma
55.025803797376.8793600128
wt
kd2
NF-kB/CyclG1
nucl 20.11/16.12
46.882444060764.5456058631
wt
kd2
NF-kB/GAPDH
citopl 20.11/16.12
8.126591533123.7439387463
wt
kd2
NF-kB/GAPDH
Citoplasma
28.655490855830.0412797958
NF-kB/CyclinG1
Nucleo
8.126591533123.7439387463
WT
KD2
28.655490855830.0412797958
WT
KD2
Densitometria lastra del 10.2.16 cp wtp29/kd2p27ntno fbsH2O2 1 mM
HoxA5meanODTubulinameanODHoxA5/Tubulina
WTp29 NT191,19229,444WTp29 NT193,80930,26397.29
WTp29 -FBS176,49214,744WTp29 -FBS196,89433,34844.21
WTp29 1mM H2O2201,42939,681WTp29 1mM H2O2196,69633,150119.70
KD2 p27 NT194,91833,170KD2 p27 NT198,75235,20694.22
KD2 p27 -FBS184,87523,127KD2 p27 -FBS201,85738,31160.37
KD2 p27 1mM H2O2190,52728,779KD2 p27 1mM H2O2198,86935,32381.47
background161,748background163,546
Densitometria lastra 17.3.16 cp del 9.3/15.3/15.3.16
HoxA5meanODTubulinameanODHoxA5/Tubulina
wt 9.3.16174,67032,439wt 9.3.16199,48757,23256.68
kd2 9.3.16168,10325,872kd2 9.3.16212,73270,47736.71
wt 15.3.16174,33632,105wt 15.3.16222,92480,66939.80
kd2 15.3.16162,44920,218kd2 15.3.16224,14181,88624.69
wt 15.3.16161,07018,839wt 15.3.16200,07157,81632.58
kd2 15.3.16157,51715,286kd2 15.3.16203,61461,35924.91
background142,231background142,2550
Densitometria lastra 17.3.16 cp del 15.2/22.2/26.2/29.2
HoxA5meanODTubulinameanODHoxA5/Tubulina
wt 15.2.16177,71527,633wt 15.2.16182,42335,09478.74
wt 22.2.16170,40420,322wt 22.2.16187,10539,77651.09
wt 26.2.16172,95222,870wt 26.2.16181,64934,32066.64
wt 29.2.16200,54850,466wt 29.2.16188,65041,321122.13
kd2 15.2.16178,10028,018kd2 15.2.16188,68941,36067.74
kd2 22.2.16166,69516,613kd2 22.2.16197,00749,67833.44
kd2 26.2.16164,91914,837kd2 26.2.16190,93143,60234.03
kd2 29.2.16194,99044,908kd2 29.2.16172,27224,943180.04
background150,082background147,329
Densitometria lastra 20.4.16 cp del 15.4.16
HoxA5meanODTubulinameanODHoxA5/Tubulina
wt p17 1198,61841,726wt p17 1198,70544,50993.75
wt p17 2201,13044,238wt p17 2183,02128,825153.47
kd2 p19 1190,23533,343kd2 p19 1202,33348,13769.27
kd2 p19 2184,29327,401kd2 p19 2196,19041,99465.25
kd2 p19 3181,14724,255kd2 p19 3188,35034,15471.02
background156,8920background154,196
Densitometria lastra 29.4.16 cp del 21.4.16
HoxA5meanODTubulinameanODHoxA5/Tubulina
WT 6.4.16100,62413,499WT 6.4.16131,21919,84268.03
WT p19 NS1111,41624,291WT NS1135,41824,041101.04
WT p19 S1120,45533,330WT S1142,38231,005107.50meandev.sttest t
KD2 14.4.16114,28527,160KD2 14.4.16155,93044,55360.96wt92.1921.16915840950.0950304527
KD2 p22 NS1115,79128,666KD2 NS1147,03635,65980.39kd270.999.72899218
KD2 p22 S1117,37830,253KD2 S1153,62542,24871.61
background87,125background111,377
nsns1s1
wt
kd2
wt
kd2
wt
kd2
wt
kd2
wt
kd2
Densitometria lastra 10.2.16 cp del 5.2.16NTNO FBSH2O2 1mM
PARPmeanODTubulinameanODPARP/Tubulina
WTp29 NT161,18514,933WTp29 NT193,80930,26349.34WT NTWT -FBSWT H2O2KD2 NTKD2 -FBSKD2 H2O2
WTp29 -FBS184,09937,847WTp29 -FBS196,89433,348113.4910.2.1649.3440835343113.491063931997.20965309224.333920354523.027328965627.71848370751mM H2O2
WTp29 1mM H2O2178,47732,225WTp29 1mM H2O2196,69633,15097.2112-18.2.1613.8809773647107.938595314119.37581274384.05015400291mM H2O2
KD2 p27 NT154,8198,567KD2 p27 NT198,75235,20624.3328.4.1614.2618.2422.229.949.407.41100uM H2O2
KD2 p27 -FBS155,0748,822KD2 p27 -FBS201,85738,31123.038.4.1615.636904208986.0782141944113.5084896238-2.167139376100uM H2O2
KD2 p27 1mM H2O2156,0439,791KD2 p27 1mM H2O2198,86935,32327.72
background146,252background163,546
mean14.9552.1667.863.899.407.41
dev.st0.97322879347.970581526864.55251334598.561717336500
Densitometria lastra 17.3.16 cp del 12-18.2.16test.t
PARPmeanODTubulinameanODPARP/Tubulina
WT NT 12.2152,4464,630WT NT 12.2149,68233,35513.88
KD2 NT 12.2149,4861,670KD2 NT 12.2157,56041,2334.05
WT 1mM H2O2 O.N. 12.2153,3295,513WT 1mM H2O2 O.N. 12.2144,78028,45319.38
WT -FBS 12.2188,03540,219WT -FBS 12.2153,58837,261107.94
WT 2.5mM H2O2 5h 12.2155,7217,905WT 2.5mM H2O2 5h 12.2155,49939,17220.18
WT NT 18.2183,18835,372WT NT 18.2151,71335,38699.96
KD2 NT 18.2151,0013,185KD2 NT 18.2148,14131,81410.01
WT -FBS 18.2173,34025,524WT -FBS 18.2138,33822,011115.96
WT 1mM H2O2 5h 18.2156,8539,037WT 1mM H2O2 5h 18.2132,01915,69257.59
KD2 -FBS 18.2145,082-2,734KD2 -FBS 18.2124,4768,149-33.55
background147,816background116,327
nb:normalizzatore no buono in ultimi pozzetti
Densitometria lastra 28.4.16 cp del 15.4.16stimolo con H2O2 100uM 48hstarvation 24h
acquisizione banda forma clivata
PARPmeanODTubulinameanODPARP/Tubulina
WT NT150,9918,015WT NT194,71256,20414.26
WT-FBS153,83110,855WT-FBS198,02859,52018.24
WT H2O2154,87311,897WT H2O2192,05653,54822.22
KD2 NT148,4485,472KD2 NT193,55355,0459.94
KD2-FBS148,7785,802KD2-FBS200,21161,7039.40
KD2 H2O2147,4544,478KD2 H2O2198,90060,3927.41
background142,976background138,508
NTNO FBSH2O2 100µM
Densitometria lastra 8.4.16 cp del 5.4.16
PARPmeanODTubulinameanODPARP/Tubulina
WT NT p26164,8586,732WT NT p26200,32543,05215.64
KD2 NTp25157,041-1,085KD2 NTp25207,33950,066-2.17
WT 100 Um H2O2 48h199,03940,913WT 100 Um H2O2 48h193,31736,044113.51
KD2 100 Um H2O2 48h160,2412,115KD2 100 Um H2O2 48h203,75146,4784.55
WT 5mM H2O2160,9692,843WT 5mM H2O2183,49226,21910.84
WT -FBS178,33220,206WT -FBS180,74723,47486.08
background158,126background157,273
wt
kd2
wt
kd2
Densitometria lastra 28.4.16 cp delnss
VEGFR2meanODTubulinameanODVEGFR2/Tubulinamean
WT NS 1133,85583,448WT NS 1156,91750,229166.14
WT NS 2142,33391,926WT NS 2163,44156,753161.98164.06
WT S1105,05654,649WT S1150,48243,794124.79
WT S296,16245,755WT S2144,05337,365122.45123.62
KD2 NS1134,93384,526KD2 NS1151,50444,816188.61
KD2 NS2136,65286,245KD2 NS2153,07746,389185.92187.26
KD2 S1102,99952,592KD2 S1157,81651,128102.86
KD2 S295,26844,861KD2 S2170,95064,26269.8186.34
background50,407background106,688
nota: NS=non starvate S=starvate
wt
kd2
Grafico1
52.930042288795.553699555728.655490855830.0412797958
WT
KD2
p-Akt
Densitometria lastra del 3-7-15 cp 1-7-15nota: bande bianche
p-AktmeanODtubulinameanODpAkt/tubwt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50
wt -ins154,7141,986wt -ins219,80756,3653.5234631427/3/153.5273.5941.2517.99144.3816.62nota: stim vegf 150
wt +ins185,22332,495wt +ins207,60044,15873.588024826/26/1523.4952.1931.4818.7156.848.39
wt +VEGF150172,02519,297wt +VEGF150210,21946,77741.25317998166/26/1515.5362.3931.6028.1690.2522.60
kd -ins162,5769,848kd -ins218,18754,74517.98885742995/7/1514.3865.9413.8914.2591.6424.78
kd +ins210,24157,513kd +ins203,27639,834144.3816839886
kd +VEGF150160,5617,833kd +VEGF150210,57147,12916.6203399181mean14.1862.7234.7821.6297.1615.87
background152,728163,442dv.st10.0510.705.615.6744.187.13
0.020.17-insVEGF 50ng/ml
lastre del 26-6-15 cp 18-6 p100
p-AktmeanODTubulinameanODpAkt/tub
wt -ins181,77516,039wt -ins237,56368,28023.4900410076
wt +ins199,80134,065wt +ins234,55165,26852.1924986211
wt +VEGF50185,31019,574wt +VEGF50231,46162,17831.4805879893
kd -ins178,52012,784kd -ins237,62468,34118.7061939392
kd +ins203,90338,167kd +ins236,43367,15056.8384214445
kd +VEGF50171,3775,641kd +VEGF50236,48467,2018.3942203241
background165,736background169,283
lastre del 26-6-15 cp 24-6 p60
p-AktmeanODTubulinameanODpAkt/tub
wt -ins176,62010,706wt -ins229,65968,92315.5332762648
wt +ins208,24842,334wt +ins228,59467,85862.3861593327
wt +VEGF50186,23520,321wt +VEGF50225,04664,31031.5985072306
kd -ins182,98117,067kd -ins221,35460,61828.1550034643
kd +ins219,01653,102kd +ins219,57658,84090.2481305235
kd +VEGF50180,01914,105kd +VEGF50223,15962,42322.5958380725
background165,914background160,736
lastre del 13-5-15 cp 8-5
p-AktmeanODMANCA NORMALIZZATORE
wt -ins97,1497,367
wt +ins131,32041,538
wt +VEGF50100,46610,684
wt +VEGF15097,0397,257
kd -ins95,7095,927
kd +ins141,01951,237
kd +VEGF5094,4204,638
kd +VEGF15094,0214,239
background89,782
lastre del 7-5-15 cp 30-4 P100
p-AktmeanODTubulinameanODpAkt/tub
wt -ins73,05011,771wt -ins146,94781,86614.38
wt +ins134,51673,237wt +ins176,153111,07265.94
wt +VEGF5078,20716,928wt +VEGF50186,950121,86913.89
kd -ins78,14816,869kd -ins183,419118,33814.25
kd +ins162,430101,151kd +ins175,455110,37491.64
kd +VEGF5073,48412,205kd +VEGF50114,32749,24624.78
background61,279background65,081
lastre del 25-9-15
IKBαmeanODTubulinameanODIKBα/tubulinameandev.sttest.t
wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622
wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30
wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39
kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474
kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33
kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82
background146,742background160,713
p-Akt
10.05161635495.6736980636
10.701721438144.1785639731
5.60847818227.1304694614
wt
kd
signaling pAKT
10.05161635495.6736980636
5.60847818227.1304694614
wt
kd
pAkt/tubulina (OD)
signaling eNOS
22.683367862232.4451951155
wt
kd
IKBa-NFkB
Densitometria 28.10.15 cp wt p25/kd2 p22-insVEGF 50ng/ml
p-AktmeanOD14.3.3meanODpAkt/14.3.3
wt p25 -176,35833,572wt p25 -191,06834,05798.5759168453wt -inswt +inswt +VEGF50kd -inskd +inskd +VEGF50wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50
wt p25 +VEGF50181,72538,939wt p25 +VEGF50189,30232,291120.58777987677/3/156.7076.9412.927.8760.384.74nota: stim vegf 1507/3/151001148.8988903168117.5298069394901.66532258917/3/15100192.9469563869117.529806939470.7924584787
kd2 p22 -151,9759,189kd2 p22 -193,27336,26225.34057691256/26/1530.1874.3140.4722.3982.248.356/26/15100246.208369934174.1862488553272.45712563726/26/15100134.071546402974.186248855327.6542550773
kd2 p22 +VEGF50154,26611,480kd2 p22 +VEGF50193,07436,06331.83318082246/26/1517.7468.4437.5834.65104.9826.826/26/15100385.8082919831195.3163440198591.7988036876/26/15100211.8245924718195.3163440198151.1999742335
background142,786background157,0115/7/1516.3674.0114.1814.6197.3224.285/7/15100452.295768442489.2992749743594.777361807810/28/15100122.329858788925.706661143532.2930608623
14.3.310/28/1598.58120.5925.3431.8312/15/15100140.721001484724.666707594812.0123648366
Densitometria lastra del 3-7-15 cp 1-7-15nota: bande nere12/15/1589.65126.1622.1110.771/13/16100593.1357814234765.8291967053736.621379007
p-AktmeanODtubulinameanODpAkt/tub96.68100.1121.808.581/18/16100186.7782247738197.0159635832185.7824134586
wt -ins157,1243,440wt -ins212,98851,3686.69677620311/13/1620.74122.99158.80152.75-
wt +ins184,17530,491wt +ins201,25039,63076.939187484275.4868.69
wt +VEGF150159,6125,928wt +VEGF150207,49845,87812.9212258599138.78108.90mean100.00558.30119.08590.17mean100.00225.97200.04173.77
kd -ins157,2443,560kd -ins206,85145,2317.87070814261/18/1616.3530.5432.2230.38dv.st0.00402.9953.90256.90dv.st0.00165.37259.47256.77
kd +ins187,30833,624kd +ins217,30555,68560.38250875460.030.0060.030.426
kd +VEGF150155,7392,055kd +VEGF150204,96743,3474.740812513mean58.2072.2573.1823.4894.8518.440.2530.164
background153,684161,620dv.st40.683.3148.166.5211.5710.41
0.290.17
lastre del 26-6-15 cp 18-6 p100nota: in rosso esclusi esperimenti wt no risp a VEGF
p-AktmeanODTubulinameanODpAkt/tub
wt -ins185,66718,099wt -ins226,02759,96430.1831098659
wt +ins209,45141,883wt +ins222,42356,36074.3133427963
wt +VEGF50189,38921,821wt +VEGF50219,98653,92340.4669621497
kd -ins180,54412,976kd -ins224,01357,95022.3917169974
kd +ins214,40846,840kd +ins223,02156,95882.2360335686
kd +VEGF50172,2804,712kd +VEGF50222,51556,4528.3469141926
background167,568background166,063
lastre del 26-6-15 cp 24-6 p60
p-AktmeanODTubulinameanODpAkt/tub
wt -ins176,0819,811wt -ins222,00055,30617.7394857701
wt +ins203,86537,595wt +ins221,62554,93168.4404070561
wt +VEGF50185,34219,072wt +VEGF50217,44950,75537.5765934391
kd -ins182,55616,286kd -ins213,69847,00434.648115054wt -inswt +inskd -inskd +inswt -inswt +VEGF50kd -inskd +VEGF50
kd +ins213,68247,412kd +ins211,85645,162104.98206456767/3/156.7076.947.8760.387/3/156.7012.927.874.74
kd +VEGF50179,40813,138kd +VEGF50215,67648,98226.82209791356/26/1530.1874.3122.3982.246/26/156.048.094.481.67
background166,270background166,6946/26/1517.7468.4434.65104.986/26/157.1015.0313.8610.73
5/7/1516.3674.0114.6197.3210/28/156.167.541.581.99
lastre del 13-5-15 cp 8-512/15/155.988.411.470.72
p-AktmeanOD1/13/166.9141.0052.9350.92
wt -ins97,4765,6901/18/165.4510.1810.7410.13
wt +ins140,53548,749
wt +VEGF50101,7709,984
wt +VEGF15097,8466,060mean17.7573.4319.8886.23mean6.3314.7413.2811.56
kd -ins96,2714,485dv.st9.643.5711.4919.65dv.st0.5911.9018.0817.82
kd +ins152,95761,1710.000.0010.040.430
kd +VEGF5094,9893,2030.3930.165
kd +VEGF15094,2292,443
background91,786
lastre del 7-5-15 cp 30-4 P100
p-AktmeanODTubulinameanODpAkt/tub
wt -ins77,72512,031wt -ins137,56873,52516.36
wt +ins140,32474,630wt +ins164,881100,83874.01
wt +VEGF5081,29715,603wt +VEGF50174,046110,00314.18
kd -ins81,30915,615kd -ins170,906106,86314.61
kd +ins162,26496,570kd +ins163,26899,22597.32
kd +VEGF5076,37010,676kd +VEGF50108,00743,96424.28
background65,694background64,043
lastre del 15.12.15 cp del 3.12.15 (Potito) wtp19/kd2p17
p-AktmeanODActinameanODpAkt/actin
wt -1113,69431,230wt -1150,46634,83589.65
wt -2116,89134,427wt -2151,23935,60896.68
wt +VEGF50 1126,88344,419wt +VEGF50 1150,84035,209126.16
wt +VEGF50 2121,28538,821wt +VEGF50 2154,41138,780100.11
kd2 -192,77210,308kd2 -1162,24446,61322.11
kd2 -296,53114,067kd2 -2180,15464,52321.80
kd2 +VEGF50 188,0715,607kd2 +VEGF50 1167,69652,06510.77
kd2 +VEGF50 284,9332,469kd2 +VEGF50 2144,41728,7868.58
background82,464background115,631
TubulinameanODpAkt/tub
wt -1200,53970,57444.25
wt -2215,51085,54540.24
wt +VEGF50 1216,74786,78251.18
wt +VEGF50 2218,93188,96643.64
kd2 -1213,24283,27712.38
kd2 -2213,29483,32916.88
kd2 +VEGF50 1215,84185,8766.53
kd2 +VEGF50 2208,25178,2863.15
background129,965
lasta del 13-1-16 cp del 17.12.15
p-AktmeanOD14.3.3meanODpAkt/14.3.3
wt -1102,57910,637wt -1149,25551,29720.74
wt +VEGF50 1157,64565,703wt +VEGF50 1151,37853,420122.99
wt -2146,85754,915wt -2170,71672,75875.48
wt +VEGF50 2127,43335,491wt +VEGF50 2149,62951,67168.69
kd2 -1167,57275,630kd2 -1145,58347,625158.80
kd2 +VEGF50 3187,56995,627kd2 +VEGF50 3160,56362,605152.75
wt -3168,06476,122wt -3152,80854,850138.78
wt +VEGF50 3147,39555,453wt +VEGF50 3148,87950,921108.90
background91,942background97,958
lastra del 20.1.16 wb 18.1.16 cp vedi quaderno
p-AktmeanODTubulinameanODpAkt/tubulina
wt p20 -2182,78811,443wt p20 -2207,50869,97416.35
wt p20 +VEGF50 2192,43021,085wt p20 +VEGF50 2206,56569,03130.54
kd2 p18 -1192,30620,961kd2 p18 -1202,59365,05932.22
kd2 p18 +VEGF50 1190,17618,831kd2 p18 +VEGF50 1199,51661,98230.38
background171,345background137,534
IKBa-NFkB
40.67599330046.5221844176
3.306600167811.5735223607
48.161030696710.4093376061
wt
kd
pAKT/Tub
NF-kB n-c
053.9033817395
402.9882219996256.9012048943
wt
kd2
HoxA5
0259.468648097
165.3739785067256.7718898938
wt
kd2
PARP
Densitometria 28.10.15 cp wt p25/kd2 p22
eNOSmeanOD14.3.3meanODeNOS/14.3.3
wt p25 -172,37030,350wt p25 -191,06834,05789.1153066917
wt p25 +VEGF50176,62634,606wt p25 +VEGF50189,30232,291107.1691802669
kd2 p22 -162,22020,200kd2 p22 -193,27336,26255.7056974243
kd2 p22 +VEGF50161,48219,462kd2 p22 +VEGF50193,07436,06353.9666694396
background142,020background157,011
VEGFR2
Densitometria lastra del 21-10-15 cp del 19-10-15nota: bande nere
lastre del 25-9-15
IKBαmeanODTubulinameanODIKBα/tubulinameandev.sttest.t
wt p34 9-7192,68345,941wt p34 9-7200,69839,985114.9090.1922.6833678622
wt 2sc p29 7-4174,96628,224wt 2sc p29 7-4200,86140,14870.30
wt p29 15-7185,34238,600wt p29 15-7205,92045,20785.39
kd p34 9-7165,48118,739kd p34 9-7196,44435,73152.4484.8732.44519511550.4135791474
kd 2sc p29 7-4186,24439,502kd 2sc p29 7-4194,37933,666117.33
kd p29 15-7177,21830,476kd p29 15-7196,64335,93084.82
background146,742background160,713
lastra del 25-11-15lastra del 21-10
NF-kB p65meanOD14.3.3meanODNF-kB p65/14.3.3
wt -114,57936,699wt -144,06662,96558.28
wt +v50151,12973,249wt +v50138,67757,576127.22
kd -147,83569,955kd -158,98677,88589.82
kd +v50180,424102,544kd +v50179,32398,222104.40
kd2 -144,75166,871kd2 -174,70193,60071.44
kd2 +v50171,70393,823kd2 +v50151,83770,736132.64
background77,880background81,101
-VEGF50
lastra del 21.10
IKB-αmeanODIKB-α/14.3.3
wt -138,70058,30592.60
wt +v50146,43066,035114.69
kd -162,14481,749104.96
kd +v50172,73892,34394.01
kd2 -167,39987,00492.95
kd2 +v50147,05666,66194.24
background80,395
VEGFR2
90.193494332384.866712302922.683367862232.4451951155
wt
kd
wt
kd
kd2
NF-kB p65
wt
kd
kd2
IKB-α
Densitometria lastra del 20-11-15 cp estrazione nucleo citosol: wtp17/kd2p15 (1x106 cell)+ wtp18/kdp18 (5x105 cell)nota: bande nerecitoplnuclSUMM31.748144877837.0358623023
NF-KbmeanODGAPDHmeanODNF-kB/GAPDHActinameanODNF-kB/ActinacitoplasmaNF-kB/GAPDH
wt p17 citopl130,44366,272wt p17 citopl191,99949,096134.98wt p17 citopl90,62918,472358.77citoplasmaNF-kB/GAPDH
kd2 p15 citopl171,805107,634kd2 p15 citopl196,12353,220202.24kd2 p15 citopl119,69647,539226.41WTKD2fold
wt p18 citopl168,943104,772wt p18 citopl182,82200.00wt p18 citopl118,86946,712224.29esp 20.11134.98202.241.4982960439WTKD2
kd p18 citopl169,506105,335kd p18 citopl175,08800.00kd p18 citopl132,44660,289174.72esp 13.1.1649.0880.761.6454767726esp 20.1130.993824443588.0407348343
background64,171background142,903background72,157esp.5.2.1659.48103.681.7432309455esp 16.1231.748144877837.0358623023
esp.10.2.16209.42269.311.2859996977esp 13.1.1649.0880.76
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1esp 16.12222.2370141448259.2510361161.1665520126esp.5.2.1639.650459142769.1199073797
wt p17 nucleo87,45223,281wt p17 nucleo134,51656,72141.04mean81.1785629047128.8932870232esp.10.2.1629.917247183138.4735708332
kd2 p15 nucleo101,94337,772kd2 p15 nucleo121,29343,49886.84dev st46.882444060764.5456058631
wt p18 nucleo75,05310,882altra lastrawt p18 nucleo129,04051,24521.241.5877749299mean36.277935129462.6860150699
kd p18 nucleo74,73210,561kd p18 nucleo126,27448,47921.780.1793685306dev st8.126591533123.7439387463
background64,171background77,7951.7279377905
0.0232329513
nucleoNF-kB/CyclinG1
nucleoNF-kB/CyclinG1
WTKD2fold
esp 20.1141.0486.842.1159844055WTKD2
esp 16.1279.8485.241.0675814633esp 20.1127.3657.8933333333
esp 13.1.16151.0826939471247.08973322551.6354602024esp 16.1279.8485.24
esp 5.2.1628.9738430584115.53557862753.9875821235lamin Besp 13.1.1675.5413469736123.5448666128
esp.10.2.16esp 5.2.1628.9738430584115.5355786275lamin B
esp.10.2.16
mean75.2353790321133.6765828756
dev st55.025803797376.8793600128mean52.930042288795.5536995557
1.7767782205dev st28.655490855830.0412797958
0.13127368911.8052828871
0.0429221995
Densitometria lastra del 16-12-15 cp estrazione nucleo citosol: wtp20/kd2 p19 25.11 /kd2 p20 1.12nota: kd2 errore CERII
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p20 citopl172,08893,484wt p20 citopl190,74242,065222.24
kd2 p19 citopl140,63962,035kd2 p19 citopl158,6029,925625.04
kd2 p20 citopl218,724140,120kd2 p20 citopl202,72554,048259.25
background78,604background148,677
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1
wt p20 nucleo112,69634,612wt p20 nucleo144,31643,34979.84
kd2 p19 nucleo115,76937,685kd2 p19 nucleo147,31946,35281.30
kd2 p20 nucleo137,90759,823kd2 p20 nucleo171,14870,18185.24
background78,084background100,967
Densitometria lastra del 13.1.16 cp estrazione nucleo citosol del 11.1.16 wtp19/kd2p18
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p19 citopl127,15329,146wt p19 citopl145,80559,37949.08
kd2 p18 citopl145,84247,835kd2 p18 citopl145,65559,22980.76
background98,007background86,426
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1
wt p19 nucleo115,72917,722wt p19 nucleo162,55511,730151.08
kd2 p18 nucleo134,68536,678kd2 p18 nucleo165,66914,844247.09
background98,007background150,825
Densitometria lastra del 5.2.16 cp estrazione nucleo citosol del 1.2.16 wtp28/kd2p26
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p28 citopl185,19146,849wt p28 citopl150,35278,77059.48
kd2 p26 citopl207,59669,254kd2 p26 citopl138,37866,796103.68
background138,342background71,582
NF-KbmeanODLamin BmeanODNF-kB/Lamin B
wt p28 nucleo151,30212,960wt p28 nucleo187,60144,73028.97
kd2 p26 nucleo174,84236,500kd2 p26 nucleo174,46331,592115.54
background138,342background142,871
Densitometria lastra del 10.2.16 cp 8.2.16 estrazione nucleo citosol del wtp19/kd2p20
NF-KbmeanODGAPDHmeanODNF-kB/GAPDH
wt p19 citopl197,50344,793wt p19 citopl193,18421,389209.42
kd2 p20 citopl223,79371,083kd2 p20 citopl198,18926,394269.31
background152,710background171,795
NF-KbmeanODCyclin G1meanODNF-kB/CyclinG1
wt p19 nucleo168,89216,182wt p19 nucleo
kd2 p20 nucleo173,43320,723kd2 p20 nucleo
background152,710background
manca normalizzatore nucleo: Lamin B (lastra 17.2.16) troppo vicina a NFKB
wt p17
kd2 p15
NF-kB/Actin
wt p18
kd p18
wt p17
kd2 p15
NF-kB/Cyclin G1
wt p18
kd p18
wt
kd2
NF-kB/cyclG1
nucleo
wt
kd2
NF-kB/GAPDH
Citoplasma
55.025803797376.8793600128
wt
kd2
NF-kB/CyclG1
nucl 20.11/16.12
46.882444060764.5456058631
wt
kd2
NF-kB/GAPDH
citopl 20.11/16.12
8.126591533123.7439387463
wt
kd2
NF-kB/GAPDH
Citoplasma
28.655490855830.0412797958
NF-kB/CyclinG1
Nucleo
8.126591533123.7439387463
WT
KD2
28.655490855830.0412797958
WT
KD2
Densitometria lastra del 10.2.16 cp wtp29/kd2p27ntno fbsH2O2 1 mM
HoxA5meanODTubulinameanODHoxA5/Tubulina
WTp29 NT191,19229,444WTp29 NT193,80930,26397.29
WTp29 -FBS176,49214,744WTp29 -FBS196,89433,34844.21
WTp29 1mM H2O2201,42939,681WTp29 1mM H2O2196,69633,150119.70
KD2 p27 NT194,91833,170KD2 p27 NT198,75235,20694.22
KD2 p27 -FBS184,87523,127KD2 p27 -FBS201,85738,31160.37
KD2 p27 1mM H2O2190,52728,779KD2 p27 1mM H2O2198,86935,32381.47
background161,748background163,546
Densitometria lastra 17.3.16 cp del 9.3/15.3/15.3.16
HoxA5meanODTubulinameanODHoxA5/Tubulina
wt 9.3.16174,67032,439wt 9.3.16199,48757,23256.68
kd2 9.3.16168,10325,872kd2 9.3.16212,73270,47736.71
wt 15.3.16174,33632,105wt 15.3.16222,92480,66939.80
kd2 15.3.16162,44920,218kd2 15.3.16224,14181,88624.69
wt 15.3.16161,07018,839wt 15.3.16200,07157,81632.58
kd2 15.3.16157,51715,286kd2 15.3.16203,61461,35924.91
background142,231background142,2550
Densitometria lastra 17.3.16 cp del 15.2/22.2/26.2/29.2
HoxA5meanODTubulinameanODHoxA5/Tubulina
wt 15.2.16177,71527,633wt 15.2.16182,42335,09478.74
wt 22.2.16170,40420,322wt 22.2.16187,10539,77651.09
wt 26.2.16172,95222,870wt 26.2.16181,64934,32066.64
wt 29.2.16200,54850,466wt 29.2.16188,65041,321122.13
kd2 15.2.16178,10028,018kd2 15.2.16188,68941,36067.74
kd2 22.2.16166,69516,613kd2 22.2.16197,00749,67833.44
kd2 26.2.16164,91914,837kd2 26.2.16190,93143,60234.03
kd2 29.2.16194,99044,908kd2 29.2.16172,27224,943180.04
background150,082background147,329
Densitometria lastra 20.4.16 cp del 15.4.16
HoxA5meanODTubulinameanODHoxA5/Tubulina
wt p17 1198,61841,726wt p17 1198,70544,50993.75
wt p17 2201,13044,238wt p17 2183,02128,825153.47
kd2 p19 1190,23533,343kd2 p19 1202,33348,13769.27
kd2 p19 2184,29327,401kd2 p19 2196,19041,99465.25
kd2 p19 3181,14724,255kd2 p19 3188,35034,15471.02
background156,8920background154,196
Densitometria lastra 29.4.16 cp del 21.4.16
HoxA5meanODTubulinameanODHoxA5/Tubulina
WT 6.4.16100,62413,499WT 6.4.16131,21919,84268.03
WT p19 NS1111,41624,291WT NS1135,41824,041101.04
WT p19 S1120,45533,330WT S1142,38231,005107.50meandev.sttest t
KD2 14.4.16114,28527,160KD2 14.4.16155,93044,55360.96wt92.1921.16915840950.0950304527
KD2 p22 NS1115,79128,666KD2 NS1147,03635,65980.39kd270.999.72899218
KD2 p22 S1117,37830,253KD2 S1153,62542,24871.61
background87,125background111,377
nsns1s1
wt
kd2
wt
kd2
wt
kd2
wt
kd2
wt
kd2
Densitometria lastra 10.2.16 cp del 5.2.16NTNO FBSH2O2 1mM
PARPmeanODTubulinameanODPARP/Tubulina
WTp29 NT161,18514,933WTp29 NT193,80930,26349.34WT NTWT -FBSWT H2O2KD2 NTKD2 -FBSKD2 H2O2
WTp29 -FBS184,09937,847WTp29 -FBS196,89433,348113.4910.2.1649.3440835343113.491063931997.20965309224.333920354523.027328965627.71848370751mM H2O2
WTp29 1mM H2O2178,47732,225WTp29 1mM H2O2196,69633,15097.2112-18.2.1613.8809773647107.938595314119.37581274384.05015400291mM H2O2
KD2 p27 NT154,8198,567KD2 p27 NT198,75235,20624.3328.4.1614.2618.2422.229.949.407.41100uM H2O2
KD2 p27 -FBS155,0748,822KD2 p27 -FBS201,85738,31123.038.4.1615.636904208986.0782141944113.5084896238-2.167139376100uM H2O2
KD2 p27 1mM H2O2156,0439,791KD2 p27 1mM H2O2198,86935,32327.72
background146,252background163,546
mean14.9552.1667.863.899.407.41
dev.st0.97322879347.970581526864.55251334598.561717336500
Densitometria lastra 17.3.16 cp del 12-18.2.16test.t
PARPmeanODTubulinameanODPARP/Tubulina
WT NT 12.2152,4464,630WT NT 12.2149,68233,35513.88
KD2 NT 12.2149,4861,670KD2 NT 12.2157,56041,2334.05
WT 1mM H2O2 O.N. 12.2153,3295,513WT 1mM H2O2 O.N. 12.2144,78028,45319.38
WT -FBS 12.2188,03540,219WT -FBS 12.2153,58837,261107.94
WT 2.5mM H2O2 5h 12.2155,7217,905WT 2.5mM H2O2 5h 12.2155,49939,17220.18
WT NT 18.2183,18835,372WT NT 18.2151,71335,38699.96
KD2 NT 18.2151,0013,185KD2 NT 18.2148,14131,81410.01
WT -FBS 18.2173,34025,524WT -FBS 18.2138,33822,011115.96
WT 1mM H2O2 5h 18.2156,8539,037WT 1mM H2O2 5h 18.2132,01915,69257.59
KD2 -FBS 18.2145,082-2,734KD2 -FBS 18.2124,4768,149-33.55
background147,816background116,327
nb:normalizzatore no buono in ultimi pozzetti
Densitometria lastra 28.4.16 cp del 15.4.16stimolo con H2O2 100uM 48hstarvation 24h
acquisizione banda forma clivata
PARPmeanODTubulinameanODPARP/Tubulina
WT NT150,9918,015WT NT194,71256,20414.26
WT-FBS153,83110,855WT-FBS198,02859,52018.24
WT H2O2154,87311,897WT H2O2192,05653,54822.22
KD2 NT148,4485,472KD2 NT193,55355,0459.94
KD2-FBS148,7785,802KD2-FBS200,21161,7039.40
KD2 H2O2147,4544,478KD2 H2O2198,90060,3927.41
background142,976background138,508
NTNO FBSH2O2 100µM
Densitometria lastra 8.4.16 cp del 5.4.16
PARPmeanODTubulinameanODPARP/Tubulina
WT NT p26164,8586,732WT NT p26200,32543,05215.64
KD2 NTp25157,041-1,085KD2 NTp25207,33950,066-2.17
WT 100 Um H2O2 48h199,03940,913WT 100 Um H2O2 48h193,31736,044113.51
KD2 100 Um H2O2 48h160,2412,115KD2 100 Um H2O2 48h203,75146,4784.55
WT 5mM H2O2160,9692,843WT 5mM H2O2183,49226,21910.84
WT -FBS178,33220,206WT -FBS180,74723,47486.08
background158,126background157,273
wt
kd2
wt
kd2
Densitometria lastra 28.4.16 cp delnss
VEGFR2meanODTubulinameanODVEGFR2/Tubulinamean
WT NS 1133,85583,448WT NS 1156,91750,229166.14
WT NS 2142,33391,926WT NS 2163,44156,753161.98164.06
WT S1105,05654,649WT S1150,48243,794124.79
WT S296,16245,755WT S2144,05337,365122.45123.62
KD2 NS1134,93384,526KD2 NS1151,50444,816188.61
KD2 NS2136,65286,245KD2 NS2153,07746,389185.92187.26
KD2 S1102,99952,592KD2 S1157,81651,128102.86
KD2 S295,26844,861KD2 S2170,95064,26269.8186.34
background50,407background106,688
nota: NS=non starvate S=starvate
wt
kd2
-
*
0
0,1
0,2
0,3
0,4
0,5
0,6
WTMAECs
Glo1 KDMAECs
IgG
NF-
kB-p
65 (%
ofi
nput
)
0.0
0.1
0.2
0.3
0.4
0.5
0.6
NF-κB-p65 binding site:5’-TGGGCTTTTCC-3’
EV
NF-kB-p65
pGL3 NF-kB-p65 bs
- - +
**
0
1
2
3
4
5
6
7
8
9
10
Luc
ifera
se A
ctiv
ity(F
iref
ly R
LU
/Ren
illa
RL
U)
WTMAECs
Glo1KDMAECs
JSH-23 60µM - - +
Hox
A5
mR
NA
leve
ls(R
EU
)
** **
0
0,5
1
1,5
2
2,5
3
0.0
0.5
1.5
1.0
2.0
2.5
3.0
+
***
Glo1KD MAECs
NF-κB-p65 role in the regulation of MGO-mediatedHoxA5 overexpression
NF-kB-p65 positively regulates HoxA5 expression
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-
Summary 2
Glo1KD MAECs
nucleus
cytosol
HoxA5 gene HoxA5 mRNA
HoxA5
Anti-angiogenic factors
Pro-angiogenic factors
Glo1 MG
↑ NFkB
IKBα
NFkB ↑ NFkB mRNA
p50 p65
IKBα
p50 p65
p50 p65
ENDOTHELIAL CELL
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-
Conclusion
Partial deletion of Glo 1, associated to endogenous MGO accumulation, promotes
glucose intolerance, β-cell and vascular dysfunction
in a context of normglycaemia
Further efforts in the development of pharmacological intervention to interfere with
these molecular events, that lead to dicarbonyl stress, are needed to provide
new therapeutic options aimed at preventing the onset and progression of
vascular complications in diabetes
→ MGO levels and Glo 1 activity themselves are drivers of
glucose homeostasis impairment and vascular defects
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-
Aknowledgments
Fundings
Dr Claudia Miele
Prof Francesco Beguinot
Prof Pietro Formisano
Prof Peter P Nawroth
Dr Thomas Fleming
SID/FO.DI.RI (2013-2015) and EFSD/Novo Nordisk (2015-1017) to C. Miele
Dr Michele Ciccarelli
EASD Study GroupReactive Metabolites in Diabetes
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-
0
20
40
60
80
100
120
140
160
Trig
lyce
rides
(mg/
dl)
0
20
40
60
80
100
120
Chol
este
rol (
mg/
dl)
WTmice
Glo1KDmice
WTmice
Glo1KDmice
**
Diaposit
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-
WT mice
Glo1KDmice
F4/80 staining
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-
Glo1KD mice
0
20
40
60
80
100
120
140
160
180
5 10 15
Syst
olic
blo
od p
ress
ure
(mm
Hg)
Age (months)
wt mice
Glo1kd mice
**
*
Glo1KD mice
WT mice
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-
Glo1KD mice
0
0,2
0,4
0,6
0,8
1
1,2
% o
fres
idua
lvas
ocon
stric
tion
*** **
1.2
1.0
0.8
0.6
0.4
0.2
0.0
Glo1KD mice at 10 months of age show a higher systolic blood pressure and impaired endothelial-dependent vasodilation
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Diapositiva numero 1Diapositiva numero 2Diapositiva numero 3Diapositiva numero 4Diapositiva numero 5Diapositiva numero 6Diapositiva numero 7Diapositiva numero 8Diapositiva numero 9Diapositiva numero 10Diapositiva numero 11Diapositiva numero 12Diapositiva numero 13Diapositiva numero 14Diapositiva numero 15Diapositiva numero 16Diapositiva numero 17Diapositiva numero 18Diapositiva numero 19Diapositiva numero 20Diapositiva numero 21Diapositiva numero 22Diapositiva numero 23Diapositiva numero 24Diapositiva numero 25Diapositiva numero 26Diapositiva numero 27Diapositiva numero 28Diapositiva numero 29Diapositiva numero 30Diapositiva numero 31Diapositiva numero 32