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Accepted Manuscript
Influence of speed of sample processing on placental energetics and signallingpathways: implications for tissue collection
H.W. Yung, F. Colleoni, D. Atkinson, E. Cook, A.J. Murray, G.J. Burton, D.S.Charnock-Jones
PII: S0143-4004(13)00836-9
DOI: 10.1016/j.placenta.2013.11.016
Reference: YPLAC 2912
To appear in: Placenta
Received Date: 10 May 2013
Revised Date: 20 November 2013
Accepted Date: 24 November 2013
Please cite this article as: Yung H, Colleoni F, Atkinson D, Cook E, Murray A, Burton G, Charnock-Jones D, Influence of speed of sample processing on placental energetics and signalling pathways:implications for tissue collection, Placenta (2013), doi: 10.1016/j.placenta.2013.11.016.
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Influence of speed of sample processing on placental energetics and 1
signalling pathways: implications for tissue collection. 2
3
1Yung HW, 1Colleoni F, 1Atkinson D, 2,3Cook E, 1Murray AJ, 1,3Burton GJ, 4
1,2,3Charnock-Jones DS. 5
6
1 Centre for Trophoblast Research, Department of Physiology, Development and 7
Neuroscience, University of Cambridge, Downing Street, Cambridge, CB2 3EG, U.K. 8
2 Department of Obstetrics and Gynaecology, University of Cambridge, Rosie 9
Hospital, Robinson Way, Cambridge, CB2 0SW, UK. 10
3 National Institute for Health Research Cambridge Comprehensive Biomedical 11
Research Centre. 12
13
Running title: 14
15
Changes in energy status and signalling following delivery 16
17
Corresponding author: 18
Dr Hong wa Yung 19
Centre for Trophoblast Research, 20
Department of Physiology, Development and Neuroscience, 21
University of Cambridge, 22
Downing Street, Cambridge, 23
CB2 3EG, United Kingdom 24
25
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Telephone : +44 1223 333862 26
Email Address : hwy20@hermes.cam.ac.uk27
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Abstract 28
29
Introduction: The placenta is metabolically highly active due to extensive 30
endocrine and active transport functions. Hence, placental tissues soon become 31
ischaemic after separation from the maternal blood supply. Ischaemia rapidly 32
depletes intracellular ATP, and leads to activation of stress-response pathways 33
aimed at reducing metabolic demands and conserving energy resources for vital 34
functions. Therefore, this study aimed to elucidate the effects of ischaemia ex vivo 35
as may occur during tissue collection on phosphorylation of placental proteins 36
and kinases involved in growth and cell survival, and on mitochondrial 37
complexes. 38
Methods: Eight term placentas obtained from normotensive non-laboured 39
elective caesarean sections were kept at room-temperature and sampled at 10, 40
20, 30 and 45 minutes after delivery. Samples were analysed by Western 41
blotting. 42
Results: Between 10 and 45 min the survival signalling pathway intermediates, 43
P-AKT, P-GSK3α and β, P-4E-BP1 and P-p70S6K were reduced by 30 to 65%. 44
Stress signalling intermediates, P-eIF2α increased almost 3 fold after 45 min. 45
However, other endoplasmic reticulum stress markers and the Heat Shock 46
Proteins, HSP27, HSP70 and HSP90, did not change. Phosphorylation of AMPK, 47
an energy sensor, was elevated 2 fold after 45 min. Contemporaneously, there 48
was an ~25% reduction in mitochondrial complex IV subunit I. 49
Discussion and conclusions: These results suggest that for placental signalling 50
studies, samples should be taken and processed within 10 min of caesarean 51
delivery to minimize the impact of ischaemia on protein phosphorylation. 52
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Introduction 53
Placental dysfunction lies at the heart of the ‘Great Obstetrical Syndromes’, 54
including growth restriction, pre-eclampsia, pre-term delivery and stillbirth. 55
These syndromes are related to a varying degree with a deficiency in deep 56
trophoblast invasion, and subsequent remodelling of the uterine spiral arteries 57
[1]. Over the last few years, considerable progress has been made in elucidating 58
the pathophysiological changes within the placenta at the molecular level. 59
Studies have revealed increased oxidative stress, and activation of stress-60
response signalling pathways consistent with malperfusion [2-4]. Unlike 61
histological changes, which are relatively slow processes, alterations in 62
transcript abundance and activation of signalling pathways occur rapidly in 63
response to external stimuli. The placenta inevitably undergoes a period of 64
ischaemia after delivery, following separation from the maternal arterial supply. 65
Therefore, the speed of tissue sampling and processing is likely to be crucial in 66
molecular studies in order to avoid the introduction of ex vivo artefacts. 67
68
The placenta has a high rate of oxygen and glucose consumption, reflecting its 69
high metabolic activity [5-7]. This can be accounted for by the existence of 70
numerous active transport systems for maternal-fetal transfer of nutrients, and 71
its endocrine function. The majority of nutrients including amino acids, vitamins, 72
Ca2+ and other biomolecules, such as antibodies, are reliant on primary or 73
secondary active transport systems, which utilize energy either directly linked to 74
the hydrolysis of adenosine triphosphate (ATP) or provided by ion gradients 75
such as sodium, chloride, and protons [8]. In the sheep placenta, approximately 76
25% of total oxygen uptake is used to generate ATP to support cation co-77
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transport systems, such as Na+-K+ ATPase, which creates a Na+ gradient that is 78
the basis for secondary active transport of amino acids and other substances [9]. 79
80
Additionally, the placenta is one of the most active endocrine organs, 81
synthesizing and secreting large quantities of polypeptide hormones, such as 82
human chorionic gonadotrophin, human placental lactogen (hPL), and many 83
growth factors, including insulin growth factor 2 and placental growth factor. As 84
4 ATP molecules are required for a single peptide bond formation, protein 85
synthesis consumes a large fraction of cellular energy. Carter estimated that 86
approximately 30% of the total placental oxygen consumption is used for protein 87
synthesis [6]. 88
89
This high metabolic activity suggests that the placenta is likely to undergo 90
ischaemic changes rapidly following delivery, with depletion of its intracellular 91
energy reserves. Indeed, the concentrations of high-energy phosphates and other 92
cellular metabolites are reduced within 24 min of separation from the maternal 93
blood supply [10]. This reduction could have a serious impact on energy-94
dependent cellular processes, including protein synthesis, active transport and 95
ion transporters. As a result, stress-response signalling pathways aimed at 96
restoring homeostasis will be activated. A classic example is suppression of 97
mRNA translation in an ATP-dependent manner that aids survival of cells under 98
hypoxia [11, 12]. 99
100
In this study, our aim was to investigate the effects of ex vivo ischaemia, as would 101
be experienced by delayed processing of placental samples, on activation of 102
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stress response pathways and suppression of growth and proliferation 103
signalling, including the energy sensor AMP activated protein kinase (AMPK); 104
cell growth, metabolism, and stress signalling pathways, such as AKT-mTOR and 105
mitogen activated protein kinases (ERK1/2, p38 kinase and JNK); ER stress 106
response pathways; and heat shock protein family members. 107
108
Materials and Methods 109
All chemicals were from Sigma-Aldrich. Anti-IRE1α (phospho S724), anti-IRE1α, 110
anti-ATF6, anti-KDEL and anti-HSP40 were from Abcam (Cambridge, UK). Anti-111
HSP90 and anti-HSP70 were from Enzo Life Science (Exeter, UK). Anti-GRP78 112
was from Transduction Laboratories (BD Biosciences, Oxford, UK), and anti-β-113
actin was from Sigma-Aldrich. Other antibodies were purchased from Cell 114
Signalling Technology (New England BioLabs, Hitchin, UK). 115
116
Tissue Collection 117
The study was approved by the Cambridge Local Research Ethics Committee and 118
all participants gave written informed consent. Eight human term (38 to 40 119
weeks) placentas were obtained from normal uncomplicated singleton first 120
pregnancies after elective non-laboured caesarean section. 121
122
Separation of the placenta from the uterus was designated as t=0min. As it is 123
standard procedure in our hospital for the midwife to check placental integrity, 124
the earliest time the placenta was accessible was between 5 and 10 min after 125
separation. Therefore, for consistency, the placentas were kept at room 126
temperature and repetitively sampled at 10, 20, 30 and 45 min after separation. 127
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To minimize possible contamination with maternal decidual tissue, the surface of 128
the basal plate was removed with scissors to a depth of approximately 1-2 mm. 129
To avoid regional variations, all samples were taken from the same placental 130
lobule. At each time-point approximately a 1 cm3 piece of villous tissue was 131
taken from the selected lobule. This was rinsed twice in ice-cold PBS, and 132
several small pieces (~10 mg and ~50 mg) were quickly cut off and further 133
washed in cold PBS. These were blotted dry and snap frozen in liquid nitrogen. 134
This post-sampling procedure took approximately 2 minutes. All tissues were 135
stored at -80°C freezer for further analysis. 136
137
Western Blotting 138
Details of the procedures have been previously described [13]. Briefly, a tissue 139
lysate, was prepared using Matix D and FastPrep Homogenizer (MP Biomedicals 140
UK, Cambridge, UK) and the Bicinchoninic Acid (BCA) used to determine protein 141
concentration. Equal amounts of protein were resolved by SDS-PAGE and 142
transferred to nitrocellulose membranes. After incubation with primary and 143
secondary antibodies, enhanced chemiluminescence (ECL, GE Healthcare, Little 144
Chalfont, UK) and X-ray film (Kodak, Hempstead, UK) were used to detect the 145
bands. Multiple exposure times were employed when necessary. Unsaturated 146
bands were scanned using HP Scanjet G4050 (HP, UK) and band intensities 147
quantified by Image J (Freeware). 148
149
Statistical analysis 150
Given the number of placentas and time points, it was necessary to run 2 gels per 151
analyte. In order to combine data across the gels, densitometric values were 152
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normalized to the mean of the 10 min sample values for all the samples run on 153
the same gel. The distribution of the normalized values was assessed using the 154
Shapiro-Wilk test. If the distribution was non-normal, seven arithmetic 155
transformations were evaluated and the optimal method for generating a normal 156
distribution selected. These tests were carried out using the statistical language 157
R (version 3.0.1). For each analyte, differences between the means of the 158
transformed data for each time point were tested using one-way ANOVA with 159
repeated measures, with the Tukey correction for multiple comparisons. These 160
analyses were carried out using Prism GraphPad version 6.0. We used the 161
method of Benjamini and Hochberg to control the false discovery rate due to 162
multiple testing when groups comprised 10 or more analytes [14]. Significance 163
levels were set at p<0.05 or adjusted p<0.05. 164
165
Results 166
167
Activation of AMPK is associated with a reduction in mitochondrial 168
complex IV subunit I protein level 169
Activation of the energy sensor AMPK [15] by phosphorylation was investigated 170
in the time series of placental samples. Although the levels of phosphorylated 171
and total AMPKα did not change significantly (Suppl. Fig. 1A), the relative ratio of 172
phosphoryated to total AMPKα (P-AMPK/AMPK) showed a 2-fold significant 173
increase (p=0.022) at the 45 min time point (Fig. 1A & C). 174
175
Mitochondria are the major source of intracellular ATP production in eukaryotic 176
cells. Therefore, accumulation of AMP, which activates AMPK, suggests that 177
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mitochondrial activity in the placentas may be compromised by ex vivo 178
ischaemia. Indeed, the electron acceptor complex IV subunit I in the electron 179
transport chain (ETC) was reduced significantly (p=0.016) at 45 min after 180
separation, although the protein level of other subunits in complexes I, II, III and 181
V were not affected (Fig. 1B & D; Suppl. Fig. 1B). Loss of subunits of the ETC 182
complexes could result in reduction of mitochondrial activity, which in turn 183
compromises intracellular energy production. 184
185
Activation of ER stress signalling 186
In response to intracellular energy depletion, stress pathways are activated in an 187
attempt to restore cellular homeostasis. Therefore, we investigated changes in 188
phosphorylation or protein level of key members of the most common stress 189
signalling pathways, including the ER stress response, MAPK stress kinases, and 190
heat shock proteins. 191
192
Protein synthesis is highly energy demanding. Therefore, we first looked at the 193
ER stress response pathway. There was a significant (p=0.0075) and incremental 194
elevation of phosphorylation of eukaryotic initiation factor 2 subunit alpha (P-195
eIF2α); ~1.5 fold by 30 min, and almost 3 fold by 45 min compared to either 20 196
and 10 min respectively (Fig. 2A). However, other ER stress markers, including 197
phosphorylation of IRE1α and the levels of ATF6, GRP78 and GRP94, were 198
relatively constant (Fig. 2A; Suppl. Fig. 2A). 199
200
Next, we examined MAPK stress kinases and HSPs pathways. In the MAPK family, 201
the relative ratios between phosphorylated and total proteins of two JNK 202
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isoforms, p54 and p46, exhibited a significant reduction (p54, p=0.033 and p46, 203
p=0.02) following delayed processing (Fig. 2B). Another MAPK, p38 kinase, did 204
not change (Suppl. Fig. 2B). Cytosolic heat shock proteins P-HSP27, HSP27, 205
HSP70 and HSP90 also remained relatively constant throughout the 45 min 206
period studied (Suppl. Fig. 2C). 207
208
These data confirm that specific stress-response pathways are activated and 209
change progressively in placental tissues as time elapses after separation. 210
211
Rapid loss of AKT-mTOR signalling 212
Signalling pathways regulating cell growth and metabolism are sensitive to 213
cellular energy levels. We therefore examined the phosphorylation status of 214
kinases and proteins in the AKT-mTOR pathway, a central regulatory pathway 215
for cell growth and metabolism. Full activation of AKT is reliant on 216
phosphorylation of residues at both Ser473 and Thr308 [16]. Interestingly, 217
phosphorylation of AKT at Ser473 was reduced significantly throughout the 218
period (p=0.006), and by 60% and 65% at 30 min and 45 min respectively. In 219
contrast, phosphorylation of the Thr308 residue, which is phosphorylated by 220
upstream PI-3K-PDK1 signalling [17], was relatively constant compared to the 221
10 min control (Fig. 3; Suppl. Fig. 3). 222
223
Glycogen synthase kinase 3 (GSK-3) is a downstream target of the AKT pathway 224
whose activity is inhibited by AKT-mediated phosphorylation at Ser21 of GSK-3α 225
and Ser9 of GSK-3β. The loss of AKT phosphorylation was associated with ~30% 226
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(p=0.014) and ~40% (p=0.006) decrease in phosphorylation of GSK-3α and β 227
respectively (Fig. 3B). 228
229
Furthermore, phosphorylation of AKT at the Ser473 residue is regulated by 230
mTOR complex 2 (mTORC2) [18, 19]. Therefore, the reduction in Ser473 231
phosphorylation might indicate a decrease in mTOR signalling. Indeed, the 232
phosphorylation level of two down-stream substrates of mTOR complex 1 233
(mTORC1), 4E-binding protein 1 (4E-BP1) and p70 S6 kinase [20], were 234
inhibited by over 60% (p=0.0056) and ~40% (p=0.0064) respectively after 45 235
min (Figs. 3A & B). The other survival kinase in the MAPK pathway, ERK, was not 236
changed after 45 min from separation (Suppl. Fig. 3). 237
238
These results indicate that the activity of the AKT/mTOR pathway was rapidly 239
suppressed by the ischaemic insult. 240
241
Discussion 242
The majority of cellular processes, such as phosphorylation, protein translation 243
and ion transporter activity, are sensitive to intracellular energy levels. Our 244
results show a reduction in phosphorylation of proteins and kinases involved in 245
AKT-mTOR signalling, including 4E-BP-1, GSK-3, and AKT. There are also 246
changes in the eIF2α and JNK stress signalling pathways with increasing 247
duration of ischaemia (Figs. 2 & 3). The changes in phosphorylation of 4E-BP1 248
and eIF2α are two key molecular mechanisms involved in the inhibition of 249
protein synthesis [21], and were observed at 30 min after separation. 250
Coincidently, the level of mitochondrial ETC complex IV subunit I was reduced 251
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by over 25% at 45 min. Further experiments, including measurement of the 252
transcript encoding complex IV subunit I and their turnover rate, will be 253
required to confirm any role of protein synthesis inhibition in the reduction of 254
that subunit. However, we recently demonstrated that increased 255
phosphorylation of eIF2α following treatment of JEG3 cells with salubrinal, a 256
specific eIF2α phosphatase inhibitor, was sufficient to down-regulate complex IV 257
subunit I, indicating a potential direct regulation of translation of those proteins 258
by the eIF2α pathway [22]. Furthermore, swelling of mitochondria and other 259
organelles has been reported at 10 min after placental separation [23], indicating 260
loss of ionic homeostasis most likely due to compromised ion transporter 261
activity. Taken together, these mitochondrial impairments could serve as a 262
positive feedback loop to further reduce cellular energy production. Therefore, 263
prolonged ischaemia (over 45 min) will eventually induce necrotic cell death due 264
to severe energy depletion, resulting in loss of cell integrity and tissue damage. 265
266
To conclude, the phosphorylation status of specific placental kinases and 267
proteins involved in cell growth, metabolism, and stress signalling is changed by 268
20 min after placental separation from the uterine wall. Our findings indicate 269
that to avoid ischaemia-induced artefacts in studies focusing on these pathways, 270
placental samples must be collected and processed rapidly following delivery, 271
preferably within 10 min. Furthermore, our previous work has shown that 272
stress-response pathways are activated during a vaginal delivery [24], and so 273
only non-laboured caesarean delivered placentas are appropriate for such 274
studies. 275
276
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Acknowledgements 277
We thank the technical and midwifery staff in the Department and Obstetrics and 278
Gynaecology and the Rosie Hospital respectively for their help in obtaining the 279
placental samples. 280
281
This work was supported by the Wellcome Trust (084804/2/08/Z). The funder 282
had no role in the design, conduct or interpretation of the results. 283
284
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Figure Legends 355
Figure 1: AMPK phosphorylation and mitochondrial ETC complexes protein level 356
following ischaemia ex vivo. Western blotting analysis was used to assess the 357
phosphorylation of AMPK and level of subunits in ETC complexes. Both β-actin 358
and Ponceau S staining were used to show equal loading among samples. A) 359
AMPK; B) Mitochondrial complexes subunits. C & D) Densitometry was used to 360
quantify band intensities. Each data point was calculated relative to the mean 361
value of the 10 min values. In the dot plot graphs, the median of the group is 362
shown, n=8. "a" and "b" denote significance p<0.05 compared to 10 and 20 min 363
respectively. C) P-AMPKα/AMPKα. D) Mitochondrial complex IV subunit I. 364
365
Figure 2: Phosphorylation of eIF2α and JNK. Upper panel, protein lysates were 366
resolved by SDS-PAGE and probed with antibodies specific for total and 367
phosphorylated kinases or proteins: ER stress markers, and MAPK family 368
members. Both β-actin and Ponceau S staining were used to show equal loading 369
among samples. Lower panel, densitometry was used to quantify band intensity. 370
Each data point was calculated relative to the mean value of the 10 min values. In 371
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the dot plot graph, the median of the group is shown, n=8. "a" and "b" denote 372
significance p<0.05 to 10 min or 20 min respectively. A) ER stress response 373
pathways; B) MAPK pathways. 374
375
Figure 3: Phosphorylation of AKT, GSK-3, 4EBP-1 and p70S6K. A) 376
Phosphorylation levels of kinases and proteins in the AKT-mTOR pathway were 377
measured by Western blot. Both β-actin and Ponceau S staining were used to 378
show equal loading among samples. B) Densitometry was used to quantify band 379
intensities. Each data point was calculated relative to the mean value of the 10 min 380
values. In the dot plot graph, the median of the group is shown, n=8. "a" and "b" 381
denote significance adjusted p<0.05 to 10 min or 20 min respectively. 382
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A.
B.
Ponceau S Staining
Time (min.)
P-‐AMPKα(Thr172)
AMPKα
β-‐ac?n
Placenta 6 10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 5 Placenta 7 Placenta 8 10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 1 Placenta 2 Placenta 3 Placenta 4
Complex I subunit NDUFB8 Complex II subunit
Complex IV subunit I
Complex III subunit Core 2
ATP synthase subunit alpha
Time (min.) Placenta 6
10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 5 Placenta 7 Placenta 8 10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 1 Placenta 2 Placenta 3 Placenta 4
β-‐ac?n
a
b
C. D.
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A. Placenta 6 10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45 Placenta 5 Placenta 7 Placenta 8
10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 1 Placenta 2 Placenta 3 Placenta 4
Ponceau S Staining
β-‐ac=n
P-‐eIF2α(Ser51)
eIF2α
P-‐IRE1α(Ser724)
GRP78
GRP94
ATF6 (p50)
IRE1α
Time (min.)
a,b a
a a
B.
P-‐JNK(Thr183/Tyr185)
Placenta 6 10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45 Placenta 5 Placenta 7 Placenta 8
10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 1 Placenta 2 Placenta 3 Placenta 4
P-‐p38(Thr180/Tyr182)
Time (min.)
JNK
p38
β-‐ac=n
a
a
b
a b
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B.
Figure 3
A.
AKT
P-‐AKT(Ser473)
GSK3β
P-‐GSK3α(Ser21)/β(Ser9)
P-‐AKT(Thr308)
P-‐ERK1/2(Thr202/Tyr204)
P-‐4EBP-‐1(Ser65)
ERK1/2
4EBP-‐1
β-‐acFn
Ponceau S Staining
P-‐p70 S6K(Thr389)
Time (min.) 10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45 Placenta 6 Placenta 5 Placenta 7 Placenta 8
10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 1 Placenta 2 Placenta 3 Placenta 4
a a
a,b
a a
a a
a
b
a
a,b a
a,b a,b
a
a a,b
a
a a,b
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPTSupplementary Figure 1
Suppl. Fig. 1: No significant differences in P-AMPKα, AMPKα and the protein levels of
other mitochondrial complexes. Densitometry was used to quantify the band intensity of P-
AMPKα, AMPKα, and mitochondrial complex I, II, III and V. The distribution of the data is
presented as a dot-plot and the median is shown. All values were normalized to the mean
value of the 10 min samples. n=8. A) AMPKα and B) Mitochondrial complexes
A.
B.
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
complex I
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
Complex V
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
Complex II
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
Complex III
TimeR
elat
ive
Rat
io
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
P-AMPKα
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
AMPKα
Time
Rel
ativ
e R
atio
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPTSupplementary Figure 2
A.
B.
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
P-IRE1α
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
ATFp50
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
IRE1α
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
GRP78
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
P-IRE1α/IRE1α
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
GRP94
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
P-JNKp54
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
JNK
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
4
P-p38
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
2
4
6
8
P-p38/p38
Time
Rel
ativ
e R
atioc
c
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
p38
Time
Rel
ativ
e R
atio
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPT
Suppl. Fig. 2: No significant changes in other ER stress markers, stress MAPK members and
HSPs in delayed collected placental tissues. Protein lysates were resolved by SDS-PAGE and
probed with antibodies specific for HSPs. Densitometry was used to quantify the band
intensity. The distribution of the data is presented as a dot-plot and the median is shown. All
values were normalized to the mean value of the 10 min samples. n=8. A )ER stress response
pathway; B) MAPK stress pathways; C) HSPs.
C.
Placenta 6
10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 5 Placenta 7 Placenta 8
10 20 30 45 10 20 30 45 10 20 30 45 10 20 30 45
Placenta 1 Placenta 2 Placenta 3 Placenta 4
β-actin
P-HSP27(Ser82)
HSP90
HSP27
HSP70
Time (min.)
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
P-HSP27
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
HSP70
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
HSP27
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
HSP90
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
P-HSP27/HSP27
Time
Rel
ativ
e R
atio
MANUSCRIP
T
ACCEPTED
ACCEPTED MANUSCRIPTSupplementary Figure 3
Suppl. Fig. 3: No significant changes in kinase total protein and the relative ratio
between phosphorylated and total protein/kinases in AKT/mTOR and MAPK-ERK
signallings. Densitometry was used to quantify the band intensity. The data are
presented as a dot-plot and the median is shown. All values were normalized to the
mean value of the 10 min samples. n=8.
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
P-AKT(T308)
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
GSK3β
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
P-ERK
Time
Rel
ativ
e R
atio
1
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
AKT
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
2.5
4EBP1
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
ERK
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0.0
0.5
1.0
1.5
2.0
P-AKT(T308)/AKT
Time
Rel
ativ
e R
atio
10 m
in
20 m
in
30 m
in
45 m
in0
1
2
3
4
P-ERK/ERK
Time
Rel
ativ
e R
atio