Supplemental Information: Material and Methods.
Human subjects
The study was approved by the ethics committee of the University Hospital of ErlangenNuremberg (approval number: 3779). Samples of AML patients and healthy donors (HD)
were collected upon patient’s informed consent and in accordance to the Declaration of
Helsinki. Peripheral blood mononuclear cells (PBMCs) and bone marrow cells (BMCs) from
a total of 25 AML patients were collected. Patients’ characteristics are detailed in
supplemental Table 1. Mesenchymal stromal cells (MSCs) were isolated from iliac crest bone
marrow aspirates from six healthy donors (approval number: 200_12) and expanded while
fulfilling uniformly minimal MSC criteria.
Cell lines
The AML cell lines KG-1, OCI-AML3, and THP-1 were obtained from DSMZ
(Braunschweig, Germany) and the human BM-derived stromal cell line HS-5 from ATTC
(Manassas, VA, USA).
Cell isolation
PBMCs and BMCs were isolated from peripheral blood and bone marrow blood respectively
using Ficoll-Paque (GE Healthcare, Piscataway Township, NJ, USA). Primary CD33+CD34+
AML cells were further purified from PBMCs or BMCs using fluorescence activated cell
sorting (FACS Astrios, Beckman Coulter, Brea, CA, USA).
Co-culture experiments
Cells were cultured in 24-well plates (Corning Inc., Corning, NY, USA) in RPMI 1640
medium (Life Technologies, Carlsbad, CA, USA) supplemented with 10% fetal calf serum
(PAN Biotech, Aidenbach, Germany), 1% L-glutamine (Life Technologies, Carlsbad, CA,
USA), and 0,4% penicillin-streptomycin (Sigma-Aldrich, St- Louis, MO, USA) under
standard conditions (37°C, 5% CO2). For co-culture experiments HS-5 cells or MSCs were
1
seeded 24 hours prior the addition of AML cells in a HS-5/MSC to AML cell ratio of 1:10.
Co-cultures were performed for 24-72 hours.
Specific cell death
The cell viability was assessed utilizing a Count & Viability Assay Kit for a Muse Cell
Analyzer (Merck Millipore, Billerica, MA, USA) according to the manufacturer’s instructions
or FACS-based with viable cells being defined as 7-AADneg/Annexin-Vneg. Proportion of
specific cell death was calculated according to following formula: specific cell death (%) =
100 × (% dead cells − % baseline dead cells) / (100% − % baseline dead cells).
Antibodies and flow cytometry (FACS)
Cells were stained using fluorochrome-coupled antibodies (Supplemental Table 2). Labeling
was performed in presence of an Fc receptor-blocking reagent (Biolegend, San Diego, CA,
USA). Dead cells were excluded from further analysis using a LIVE/DEAD Fixable Aqua
Dead Cell Stain Kit (Life Technologies). Intracellular staining (ICS) was performed using BD
Cytofix/CytopermTM reagents (BD Bioscience, Franklin Lakes, NJ, USA). For CXCL12 ICS
cells were pre-treated with GolgiPlug (BD Bioscience). For intracellular detection of
phosphorylated proteins CytofixTM and PhosflowTM Perm Buffer III (both BD Bioscience)
were used. FACS data was acquired on an FACS Canto II cytometer (BD Bioscience) and
analyzed by FlowJo version 9.5 software (Treestar, Ashland, OR, USA).
Mitochondrial membrane potential
The potentiometric dye JC-1 (Cayman Chemicals, Ann Arbor, MI, USA) was used to
determine the electrochemical potential (ΔψM) across the mitochondrial membrane. Cells
were stained for 30 min at 37°C followed by FACS analysis.
Glucose and lactic acid measurement
We determined the glucose and lactic acid concentration in cell culture supernatants using a
Super GL Compact (Hitado, Möhnesee, Germany).
2
RNA preparation and quantitative real time PCR (qPCR)
Total RNA was isolated from cell lysates (innuPREP, RNA Mini Kit, Analytic Jena, Jena,
Germany) and transcribed to cDNA (Superscript First Strand Synthesis System, Life
Technologies) via reverse transcriptase PCR using a Mastercycler Nexus (Eppendorf,
Hamburg, Germany). The messenger RNA (mRNA) levels were quantified by qPCR
(Quantitect SYBR Green PCR Kit, Qiagen, Hilden, Germany) on a Rotor Gene Q (Qiagen).
The relative gene expression was calculated by normalizing the expression of the target gene
to ß-actin using gene-specific primers. In selected experiments cDNA was subjected to a
qPCR analysis of 84 genes involved in glycolysis (RT² Profiler™ PCR Array Glucose
Metabolism, Qiagen) according to the company’s recommendations.
Small interfering (si)RNA transfection
The human BM stromal cell line HS-5 was transfected with CXCL12 siRNA purchased from
Ambion (Life Technologies) at a concentration of 10 nM using Lipofectamine 3000 (Life
Technologies). For determining the siRNA efficacy transfected and control cells and their
supernatant were, at 48 hours post transfection, collected to measure CXCL12 mRNA and
released CXCL12 protein respectively.
Detection of CXCL12
Cell culture supernatants were analyzed by a sandwich enzyme-linked immunosorbent assay
(ELISA) using monoclonal antibodies for human CXCL12 (Leinco Technologies, Inc., St.
Louis, MO, USA).
Reactive oxygen species (ROS) measurement
Mitochondria-specific ROS were semi-quantified by FACS using MitoSOX (Life
Technologies).
Antioxidant capacity
The cells’ antioxidant capacity was quantified in cell lysates using a colorimetric Antioxidant
Assay Kit (Cayman Chemical) following the manufacturer’s instructions.
3
Glutathione levels
Intracellular glutathione levels were semi-quantified by FACS using the ThiolTracker violet
dye (Life Technologies).
Glucose uptake
Influx of glucose was semi-quantified by FACS based on the cells’ incorporation of
fluorescently labeled glucose analogue 2-(N-(7-Nitrobenz-2-oxa-1,3-diazol-4-yl)Amino)-2Deoxyglucose (2-NBDG).
Extracellular flux analyses
The bioenergetic profile of AML cells was determined using an XF96e Extracellular Flux
Analyzer (Seahorse Bioscience, North Billerica, MA, USA). Cells were seeded in specialized
tissue culture plates at an optimized density of 1x105 cells/well using CELL-TAK (BD
Biosciences). All measurements were carried out in octaplicates as described in detail
previously. Glycolytic parameters were determined using the Glyco Stress Test (Seahorse
Bioscience) that assesses changes of the extracellular acidification rate (ECAR/indicative for
aerobic glycolysis) upon application of glucose (10 mM), oligomycin (1 µM), and 2-DG (100
mM). Responses of the cells’ oxygen consumption rate (OCR/indicative for oxidative
phosphorylation) towards oligomycin (1 µM), FCCP (1.5 µM), and the combination of
antimycin (3 µM) and rotenone (3 µM) were evaluated yielding basic mitochondrial
parameters (Mito Stress Test, Seahorse Bioscience). The ECAR levels upon glucose injection
reflect basal glycolysis. Blocking mitochondrial ATP production by oligomycin shifts the
energy production away from mitochondrial oxidative phosphorylation (OXPHOS) towards
aerobic glycolysis and thereby leads to an ECAR increase that reveals the cells’ maximal
glycolytic capacity. The oxygen consumption rate (OCR), which acts as a surrogate for
OXPHOS, was measured simultaneously. Oligomycin blocks complex V (F0F1-ATPase) that
couples the electron transport chain with ATP synthesis. Relative OCR reduction following
oligomycin treatment indicates the proportion of ATP production linked to respiration.
4
Reagents
Dimethyl sulfoxide (DMSO), diclofenac sodium salt, and rapamycin were purchased from
Sigma-Aldrich, AMD3100 from Merck Millipore, and Ara-C from the hospital pharmacy.
Statistical analysis
Comparisons for two groups were calculated using a paired or unpaired, two-tailed Student’s
t-test. All statistical analyses were performed using GraphPad Prism Version 5 (GraphPad
Prism Software Inc., San Diego, CA, USA).
5
Supplemental Table 1: Patients’ characteristics.
UPN
Sex
Age, Years
Cytogenetic Risk†
BM Blasts, %
698
700
735
745
749
750
773
865
953
1007
1008
1016
1022
1036
1054
pAML2
872
905
838
1070
909
1103
739
1027
856
f
f
f
f
f
m
f
m
f
m
m
f
m
m
m
m
f
m
m
m
f
m
m
m
m
64
21
64
59
18
69
39
23
49
82
43
54
21
71
63
18
47
72
20
52
73
61
54
52
61
intermediate
intermediate
adverse
favorable
favorable
favorable
adverse
favorable
favorable
favorable
intermediate
intermediate
intermediate
intermediate
intermediate
adverse
intermediate
adverse
intermediate
intermediate
favorable
favorable
intermediate
adverse
intermediate
90
80
96
30
30
30
50
80
60
45
95
95
95
95
95
95
80
90
80
95
30
95
70
90
30
†
Cytogenetic risk assessment based on Döhner H et al., Blood 2010
Abbreviations: UPN, unpersonalized patient number; m, male; f, female; BM, bone marrow
6
Supplemental Table 2: Antibody list.
Antibody
Fluorochrome Clone
Isotype
Company
CD33
PE
WM53
Mouse, IgG1, κ
BioLegend
CD33
CD34
APC
BV421
WM53
561
Mouse, IgG1, κ BioLegend
Mouse, IgG2a, κ BioLegend
CD34
CD45
PE Cy7
V500
581
HI30
Mouse, IgG1, κ
Mouse, IgG1, κ
CD45
CD184
APC
PE
HI30
12G5
Mouse, IgG1, κ BioLegend
Mouse, IgG2a, κ BioLegend
CXCL12
GLUT-1
GLUT-2
GLUT-3
GLUT-4
pmTOR (pS2448)
unconjugated
APC
PE
FITC
FITC
PE
79018
202915
199017
polyclonal
mAbcam48574
021-404
Mouse, IgG1
Mouse, IgG2B
Mouse, IgG2A
Rabbit, IgG
Mouse, IgG2B
Mouse, IgG1, κ
Leinco Technologies
R&D systems
R&D systems
Abcam
Abcam
BD Bioscience
pS6 (pS235/pS236)
p4EBP1 (pT36/pT45)
7-AAD
Annexin-V
PE
PE
PerCP Cy5.5
FITC
N7-548
M31-16
Mouse, IgG1, κ
Mouse, IgG1, κ
BD Bioscience
BD Bioscience
BioLegend
BioLegend
2-NBDG
FITC
BioLegend
BD Bioscience
Cayman Chemicals
7
Supplemental Table 3: The 25 most strongly upregulated gene transcripts of genes involved
in the regulation of enzymatic pathways of glucose and glucose metabolism in AML cells
upon stromal contact.
Rank
Gene
Fold Change Name
1
2
3
4
5
6
7
8
9
10
11
12
13
14
15
16
17
18
19
20
21
22
23
24
25
PRPS2
SDHA
PRPS1
SUCLG2
PGM3
SDHC
PGM2
GBE1
ENO1
TPI1
ENO2
PGM1
G6PC3
FH
GAPDH
GALM
SDHB
PDK1
PC
ACO1
ACLY
PDPR
HPRT1
ALDOC
RPE
21,48
16,48
10,97
10,42
9,36
6,09
5,38
2,26
2,11
2,10
2,05
2,04
1,83
1,77
1,70
1,68
1,60
1,59
1,59
1,58
1,58
1,57
1,56
1,56
1,55
Phosphoribosyl pyrophosphate synthetase 2
Succinate dehydrogenase complex, subunit A, flavoprotein (Fp)
Phosphoribosyl pyrophosphate synthetase 1
Succinate-CoA ligase, GDP-forming, beta subunit
Phosphoglucomutase 3
Succinate dehydrogenase complex, subunit C
Phosphoglucomutase 2
Glucan (1,4-alpha-), branching enzyme 1
Enolase 1, (alpha)
Triosephosphate isomerase 1
Enolase 2 (gamma, neuronal)
Phosphoglucomutase 1
Glucose 6 phosphatase, catalytic, 3
Fumarate hydratase
Glyceraldehyde-3-phosphate dehydrogenase
Galactose mutarotase (aldose 1-epimerase)
Succinate dehydrogenase complex, subunit B, iron sulfur (Ip)
Phosphoenolpyruvate carboxykinase 1 (soluble)
Pyruvate carboxylase
Aconitase 1, soluble
ATP citrate lyase
Pyruvate dehydrogenase phosphatase regulatory subunit
Hypoxanthine phosphoribosyltransferase 1
Aldolase C, fructose-bisphosphate
Ribulose-5-phosphate-3-epimerase
8
100
***
viability [%]
80
60
40
20
-H
+ SH 5
S5
0
CD33
Supplemental Figure 1: Primary CD33+CD34+ AML cells (n=16 patients) were cultured for
24 hours in presence/absence (+/-) of HS-5 cells and the AML cells’ viability assessed. The
bars represent the standard error mean. Abbreviations: ***; p-value<0.001.
CD34
gluco
ECAR [mpH/min]
150
oligo
100
50
S5
H
+
H
S5
0
+
+
H
S5
80
S5
40 60
minutes
+
20
100
0
0
0
200
H
S5
0
50
KG-1
oligo
+
50
100
gluco
300
H
S5
100
oligo
+
150
OCI-AML3
gluco
150
ECAR [mpH/min]
200
THP-1
+ HS-5
- HS-5
H
ECAR [mpH/min]
250
ECAR [mpH/min]
gl
uc
os
e
ol
ig
om
yc
in
2D
G
Supplemental Figure 2: A representative dot plot analysis showing the FACS gating strategy
for the sorting of peripheral blood- and bone marrow-derived CD33+CD34+ AML cells.
Supplemental Figure 3: The extracellular acidification rate (ECAR), which is indicative for
aerobic glycolysis was continuously measured in three human AML cell lines (THP-1, OCIAML3, and KG-1) following 24 hours of culture in presence or absence (+/-) of a human HS5 stromal cell line monolayer using an XFe96 flux analyzer. Alterations of ECAR in response
to glucose (gluco) and oligomycin (oligo) application as exemplarily shown for THP-1 (left
panel) yield basal glycolysis and glycolytic capacity. The reaction is abolished by 2-DG.
Representative data for changes of basal glycolysis and glycolytic capacity upon stromal
contact are shown for THP-1, OCI-AML3, and KG-1. The bars represent the standard error
mean.
9
150
100
50
gl
gl
ECAR [mpH/min]
200
OCI-AML3
250
150
100
50
0
20
40 60
minutes
80
+ MSC
- MSC
200
150
100
50
0
0
uc
os
e
ol
ig
om
yc
in
2D
G
uc
os
e
ol
ig
om
yc
in
2D
G
uc
os
e
ol
ig
om
yc
in
2D
G
gl
KG-1
200
ECAR [mpH/min]
ECAR [mpH/min]
THP1
250
0
0
20
40 60
minutes
80
0
20
40 60
minutes
80
Supplemental Figure 4: Extracellular acidification rate (ECAR) as continuously monitored
by an XFe96 flux analyzer is an indicator for aerobic glycolysis. Responses of ECAR towards
glucose and oligomycin yield basal glycolysis and glycolytic capacity. Glycolysis is abolished
by 2-deoxy-D-glucose (2-DG). AML cell lines (THP-1, KG-1, and OCI-AML3) were
cultured in presence/absence of primary bone marrow-derived mesenchymal stromal cells
(MSCs). In the follow, ECAR/aerobic glycolysis was assessed as displayed. The bars
represent the standard error mean.
% OCR upon oligomycin
80
pAML
***
60
40
20
100
***
80
60
40
20
0
-H
+ SH 5
S5
0
-H
+ SH 5
S5
% OCR upon oligomycin
cell lines
Supplemental Figure 5: The oxygen consumption rate (OCR) as a surrogate for oxidative
phosphorylation (OXPHOS) was assessed before (=baseline) and after oligomycin injection
in AML cell lines (THP-1, OCI-AML3, and KG-1, n=9 experiments) and in primary
CD33+CD34+ FACS-sorted AML cells (n=13 patients) cultured for 24 hours in
presence/absence of HS-5 cells using an XFe96 flux analyzer. The extent of the oligomycinmediated OCR reduction (baseline OCR is set as 100%) correlates positively with the
OXPHOS’s contribution to energy production. The bars represent the standard error mean.
Abbreviations: ***; p-value<0.001.
10
OCR [pmol/min]
150
***
100
50
-H
+ SH 5
S5
0
Supplemental Figure 6: The oxygen consumption rate (OCR) is a surrogate for the cells’
respiration/oxidative phosphorylation. OCR was measured using an XFe96 flux analyzer in
AML cell lines (THP-1, OCI-AML3, and KG-1, n=9 experiments) previously cultured in
presence/absence of HS-5 for 24 hours. The bars represent the standard error mean.
Abbreviations: ***; p-value<0.001.
15000
JC-1 PE [MFI]
MitoSOX [MFI]
3000
2000
1000
10000
5000
0
-H
+ SH 5
S5
-H
+ SH 5
S5
0
Supplemental Figure 7: Mitochondrial fitness was evaluated based on the mitochondrial
production of reactive oxygen species (ROS) and the mitochondrial membrane potential of
AML cell lines (THP-1, OCI-AML3, and KG-1, n=6-9 experiments) cultured in
presence/absence of HS-5 cells for 24 hours. Both parameters were assessed by FACS using a
probe specific for mitochondrial ROS (MitoSOX, Life Technologies) and a potentiometric
dye (JC-1, Cayman Chemicals) respectively. Semi-quantification is based on the median
fluorescence index (MFI). The bars represent the standard error mean.
11
cell lines
40
p<0.05
%al in/decrease
[2-NBDG FITC MFI]
%al in/decrease [MFI]
cell lines
150
100
50
***
30
20
10
0
G
L
G UT
L 1
G UT
L 2
G UT
LU 3
T4
0
Supplemental Figure 8: AML cell lines (cell lines, n=9 experiments) were cultured for 24
hours in presence or absence of HS-5 cells. Expression of glucose transporters (GLUT1-4)
was semi-quantified based on the according median fluorescence index (MFI) by FACS. Data
is displayed as the %al GLUT1-4 MFI in-/decrease in response to stromal contact. Glucose
uptake was semi-quantified in AML cell lines (cell lines, n=9 experiments) by FACS based
on the MFI of the incorporated fluorescent glucose analog 2-NBDG. Data is displayed as the
%al 2-NBDG MFI in-/decrease in response to stromal contact. The bars represent the
standard error mean. Abbreviations: ***; p-value<0.001.
0.4
1000
0.1
0.0
O
C
KG
1
-H
+ SH 5
S5
0
0.2
I-A
M
L3
2000
0.3
P1
3000
- HS-5
+ HS-5
TH
*
antioxidants [mM]
Thioltracker [MFI]
4000
Supplemental Figure 9: AML cells lines (THP-1, OCI-AML3, and KG-1, n=9 experiments)
were cultured in presence or absence of HS-5 cells for 24 hours. The total intracellular
glutathione content was evaluated by FACS based on the median fluorescence index (MFI) of
the Thioltracker probe (Life Technologies). Cells were also lysed and their antioxidant
content was assessed using a fluorimetric assay (Cayman Chemicals). The bars represent the
standard error mean. Abbreviations: *; p-value<0.05.
12
% specific cell death
80
- HS-5
+ HS-5
60
40
20
0
0.1 1 10 100
Ara-C [µM]
cell number
Supplemental Figure 10: Primary AML cells (n=6) were cultured in presence or absence of
HS-5 bone marrow stroma cells with increasing concentrations of Ara-C for 48 hours and
specific cell death assessed using a FACS-based approach.
UPN 1027
+ HS-5
pmTOR
+ HS-5
pS6K1
+ HS-5
p4EBP1
Supplemental Figure 11: Primary FACS-sorted CD33+CD34+ AML cells (pAML, n=5-9
patients) were cultured in presence/absence of HS-5 cells for 24 hours and afterwards
analyzed by FACS in terms of pmTOR, pS6K1, and p4EBP1 as shown in representative
histogram analysis. Abbreviations: UPN; unique patient number.
13
cell lines
1000
-H
+ SH 5
S5
0
1500
1000
500
0
3000
2000
1000
0
-H
+ SH 5
S5
2000
2000
p4EBP1 [MFI]
**
-H
+ SH 5
S5
3000
4000
*
pS6K1 [MFI]
pmTOR [MFI]
2500
UPN 865
cell number
cell number
Supplemental Figure 12: The expression of phosphorylated/activated mTOR (pmTOR) and
phosphorylated forms of its downstream molecules ribosomal protein S6 kinase 1/S6K1 and
the eukaryotic initiation factor 4E binding protein 1/4 EBP1 (pS6K1 and p4EBP1) was
evaluated by FACS based on the median fluorescence index (MFI). AML cell lines (THP-1,
OCI-AML3, and KG-1, n=9 experiments) were cultured in presence/absence of HS-5 cells for
24 hours and afterwards analyzed by FACS in terms of pmTOR, pS6K1, and p4EBP1 MFI.
The bars represent the standard error of the mean. Abbreviations: UPN; unique patient
number, *; p-value<0.05, **; p-value<0.01.
pmTOR
UPN 749
BM
PB
pS6K1
Supplemental Figure 13: Primary PB- and bone marrow (BM)-derived FACS-sorted
CD33+CD34+ AML cells from six individual patients were evaluated regarding their pmTOR
and pS6K1 as shown in representative histogram analysis. Abbreviations: UPN; unique
patient number.
14
B.
2000
1500
1000
250
+ +
+
0
HS-5
Rapa
HS-5
Rapa
+ +
+
3000
2000
1000
0
0
+ +
+
HS-5
Rapa
50
+ +
+
HS-5
Rapa
+ +
+
glycolysis
basal capacity
THP-1
untreated
+ HS-5
150
+ HS-5 + RAPA
100
50
0
40
minute
60
80
100
50
0
O
20
500
0
200
0
* *
******
100
2DG
ol
ig
o
gl
uc
os
e
D.
HS-5
Rapa
+ +
+
1000
500
0
0
HS-5
Rapa
ECAR [mpH/min]
2000
4000 *** **
1500
2-NBDG [MFI]
200
4000
* ***
C KG
I-A -1
M
TH L 3
P
O K -1
C G
I-A -1
M
TH L 3
P1
400
2500
% of stromal effect
600
* **
GLUT4 [MFI]
GLUT2 [MFI]
GLUT1 [MFI]
6000
GLUT3 [MFI]
* *
800
C.
lactic acid [mg/dl]
A.
Supplemental Figure 14: AML cells lines (THP-1, OCI-AML3, and KG-1, n=9
experiments) were cultured for 24 hours in presence or absence of HS-5 cells with/without
simultaneous mTOR inhibition by rapamycin (100 nM). Next, we evaluated the effects of a
concomitant mTOR inhibition on the AML cells’ expression of (A) glucose transporters
(GLUT1-4), (B) glucose uptake (2-NBDG) as well as (C) lactic acid concentration in the cell
culture supernatant. (D) AML cell lines from comparable, as indicated, experimental settings
were used for glycolytic flux analyses (XFe96 flux analyzer). The extracellular acidification
rate (ECAR), indicative for aerobic glycolysis, was measured continuously as displayed
paradigmatically for THP-1 cells. Changes of ECAR upon addition of glucose and
oligomycin (oligo) yield basal glycolysis and the cells’ glycolytic capacity respectively.
Aerobic glycolysis is abolished by 2-DG. The impact of rapamycin on the stroma-mediated
promotion of basal glycolysis and of glycolytic capacity is shown for THP-1, OCI-AML3,
and KG-1 cells. ECAR levels measured for cells with stromal contact but without rapamycin
treatment were set as 100%. The bars represent the standard error of the mean. Abbreviations:
*; p-value<0.05, **; p-value<0.01, ***; p-value<0.001.
15
cell count
HS-5
CXCL12
Supplemental Figure 15: The FACS analysis density plot displays the CXCL12 production
in the human bone marrow stroma cell line HS-5.
cell lines
pAML
15000
10000
CXCR4 [MFI]
CXCR4 [MFI]
**
10000
5000
*
8000
6000
4000
2000
0
+ HS H 5
S5
-H
+ SH 5
S5
0
Supplemental Figure 16: Density of CXCR4 expression shown as the median fluorescence
index (MFI) was assessed by FACS for AML cell lines (THP-1, OCI-AML3, and KG-1, n=9
experiments) and primary CD33+CD34+ AML cells (pAML, n=4 patients) cultured in
presence/absence of HS-5 for 24 hours. The bars represent the standard error of the mean.
Abbreviations: *; p-value<0.05, **; p-value<0.01.
CXCL12 [pg/ml]
6000
**
4000
2000
+
siR
N
A
0
Supplemental Figure 17: HS-5 cells transfected with CXCL12 siRNA were cultured for 24
hours upon transfection and the CXLC12 secretion assessed (as compared to non-transfected
cells) by ELISA. The bars represent the standard error of the mean. Abbreviations: *; pvalue<0.05, **; p-value<0.01, ***; p-value<0.001.
16