Influence of speed of sample processing on placental energetics and signalling pathways:...

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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http://dx.doi.org/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 : [email protected]

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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


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


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


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


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

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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.

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complex I

Time

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Complex V

Time

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Complex II

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Complex III

TimeR

elat

ive

Rat

io

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P-AMPKα

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AMPKα

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MANUSCRIP

T

ACCEPTED


A.

B.

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P-IRE1α

Time

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ATFp50

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IRE1α

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GRP78

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3

P-IRE1α/IRE1α

Time

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GRP94

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3

P-JNKp54

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3

JNK

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2

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4

P-p38

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in0

2

4

6

8

P-p38/p38

Time

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atioc

c

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p38

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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


β-actin

P-HSP27(Ser82)

HSP90

HSP27

HSP70

Time (min.)

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HSP70

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HSP27

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HSP90

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P-HSP27/HSP27

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MANUSCRIP

T

ACCEPTED


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.

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P-AKT(T308)

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GSK3β

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P-ERK

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AKT

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4EBP1

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ERK

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P-AKT(T308)/AKT

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P-ERK/ERK

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Influence of speed of sample processing on placental energetics and signalling pathways:...

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Transcript of Influence of speed of sample processing on placental energetics and signalling pathways:...