Bastien
Dalzon‡
a,
Anaelle
Torres‡
a,
Hélène
Diemer
b,
Stéphane
Ravanel
c,
Véronique
Collin-Faure
a,
Karin
Pernet-Gallay
d,
Pierre-Henri
Jouneau
e,
Jacques
Bourguignon
c,
Sarah
Cianférani
b,
Marie
Carrière
f,
Catherine
Aude-Garcia§
a and
Thierry
Rabilloud
*a
aChemistry and Biology of Metals, Univ. Grenoble Alpes, CNRS UMR5249, CEA, IRIG, CBM-ProMD, 17 Rue des Martyrs, F-38054 Grenoble Cedex 9, France. E-mail: thierry.rabilloud@cnrs.fr; Tel: +33 438 783 212
bLaboratoire de Spectrométrie de Masse BioOrganique (LSMBO), Université de Strasbourg, CNRS, IPHC UMR 7178, 67000 Strasbourg, France
cUniv. Grenoble Alpes, INRA, CNRS UMR5168, CEA, IRIG, Laboratory of Plant Cellular Physiology, Grenoble, F-38000 France
dUniv. Grenoble Alpes, Inserm U1216 Grenoble Institut des Neurosciences, 38000 Grenoble, France
eUniv. Grenoble Alpes, Modelization and Exploration of Materials, CEA-DRF-IRIG-DEPHY-MEM-LEMMA, F-38000 Grenoble, France
fUniv. Grenoble-Alpes, CEA, CNRS UMR 5819, IRIG, SyMMES, Chimie Interface Biologie pour l'Environnement, la Santé et la Toxicologie (CIBEST), F-38054 Grenoble, France
First published on 20th September 2019
Silver nanoparticles are known to strongly affect biological systems, and numerous toxicological studies have investigated their effects. Most of these studies examine the effects immediately following acute exposure. In this work, we have conducted further investigation by studying not only the acute, post-exposure response, but also the cellular response after a 72 hour-recovery-phase post exposure. As a biological model we have used macrophages, which are very important cells with respect to their role in the immune response to particulate materials. To investigate the response of macrophages to nanoparticles and their recovery post exposure, we have used a combination of proteomics and targeted experiments. These experiments provided evidence that the cellular reaction to nanoparticles, including the reaction during the recovery phase, is a very active process involving massive energy consumption. Pathways such as the oxidative stress response, central and lipid metabolism, protein production and quality control are strongly modulated during the cellular response to nanoparticles, and restoration of basic cellular homeostasis occurs during the recovery period. However, some specialized macrophage functions, such as lipopolysaccharide-induced cytokine and nitric oxide production, did not return to their basal levels even 72 hours post exposure, showing that some effects of silver nanoparticles persist even after exposure has ceased.
Environmental significanceSilver nanoparticles are known to have profound effects on living cells. Because of their widespread use, contamination is almost unavoidable. In this context, it is important not only to assess the immediate effects of silver nanoparticles on cells, but also how they recover after exposure. Using macrophages as the target cell type and a combination of proteomic and targeted experiments, we show here that the recovery phase is not just a “return to normal” condition. For example, some of the specialized macrophage functions are still not restored after this time, showing that subtle but sustained effects can occur after a single exposure. Although cell survival is not affected, such effects may impact the health status of living beings. |
Many toxicological studies have reported pro-inflammatory responses5,11,12,21 and/or immunological effects15,22 pointing to macrophages as a cell type of major interest in toxicological studies of AgNPs. This is in accordance with the important scavenging function of macrophages in many different tissues.
This work investigates the recovery of macrophages after a unique, acute, but subtoxic dose of AgNPs. Targeted experiments carried out primarily on macrophages showed that several functional effects (e.g. LPS-induced cytokine and NO production, mitochondrial transmembrane potential and phagocytosis) were altered immediately after exposure to AgNPs. However, these functions tended to return to normal after a 72 h recovery period.23 We therefore decided to get a broader view of cellular recovery using a proteomic approach.
Proteomic approaches have been used to study cellular responses to AgNPs in several models such as intestinal cells9,24,25 or liver cells.10 This allowed several important pathways modulated in response to acute exposures to AgNPs to be highlighted. These included mitochondrial proteins, pointing at potential mitochondrial dysfunction,9,10,24 and proteins involved in intermediate metabolism9,10,24 or in inflammatory responses.10 The latter findings reinforced the interest in studying macrophages, which are major players in inflammation. We therefore decided to use proteomics to study the cellular responses using a macrophage J774 cell line, directly after acute exposure and after a recovery period had lapsed. This model cell line was selected because, unlike human monocyte cell lines,26,27 mouse macrophage cell lines such as RAW264.7 and J774 do not need to be chemically differentiated into macrophages, a process that deeply alters cell physiology and causes some cell death. Furthermore, as opposed to primary macrophages derived from human blood, these mouse cell lines do not show extensive variability from one experiment to another. This explains why these cell lines are extensively used as models for testing of a wide variety of nanomaterials,28–31 including large scale projects.32–34
For determination of the useful dose, cells were seeded at 500000 cells per ml. They were treated with nanoparticles on the following day and harvested after a further 24 hours in culture.
For treatment with nanoparticles, cells were seeded in classical cell culture flasks and left for 24 hours at 37 °C for cell adhesion and confluence. For the recovery condition, cells were treated with nanoparticles on the following day. After 24 hours of exposure, the cell culture medium was removed and replaced by fresh medium. For consistency reasons, this operation was also carried out on cells used for control and for acute exposure. Another medium culture change was carried out 36 hours after the initial medium change, i.e. mid-term of the 72 hour recovery period. Finally, acute exposure was carried out for the final 24 hours and the cells were used immediately afterwards.
The cell extracts for enzyme assays were prepared by lysing the cells for 20 minutes at 0 °C in Hepes (20 mM, pH 7.5), MgCl2 (2 mM), KCl (50 mM), EGTA (1 mM), and tetradecyldimethylammonio propane sulfonate (SB 3-14) (0.15% (w/v)), followed by centrifugation at 15000g for 15 minutes to clear the extract. The protein concentration was determined by a dye-binding assay.42 The dehydrogenase or dehydrogenase-coupled activities were assayed at 500 nm using the phenazine methosulfate/iodonitrotetrazolium coupled assay.43 The enzyme assay buffer contained 25 mM Hepes, NaOH (pH 7.5), 5 mM magnesium acetate, 100 mM potassium nitrate and 1% Triton X-100. It also contained 30 μM phenazine methosulfate, 200 μM iodonitrotetrazolium chloride, 250 μM of the adequate cofactor (NAD or NADP) and 1–5 mM of the organic substrate, which was used to start the reaction. For phosphate-dependent enzymes such as glyceraldehyde dehydrogenase (GAPDH) and purine phosphorylase (PNPH), 50 mM potassium phosphate (pH 7.5) was added to the enzyme assay buffer. Triose phosphate isomerase was assayed with dihydroxyacetone phosphate and a glyceraldehyde dehydrogenase-coupled assay.44 Purine phosphorylase (PNPH) was assayed by a xanthine oxidase-coupled assay.45 Hexokinase was assayed by a glucose phosphate dehydrogenase (G6PDH)-coupled assay.46 Biliverdine reductase was assayed at 450 nm as described.47 Pyridoxal kinase was assayed directly at 388 nm.48 Enolase was assayed at 340 nm by a pyruvate kinase–lactate dehydrogenase-coupled assay.49
The NADP–NADPH concentration was determined using an adapted alkaline extraction buffer.51 Briefly, at the end of the exposure period, cells were collected by scraping, rinsed twice in PBS and pelleted. The packed cell pellet (PCP) volume was estimated, and the cells were lysed in 10 PCP volumes of 10 mM CAPS, 1 mM EGTA and 2 mM MgCl2 for 10 minutes on ice with occasional vortexing. The suspension was centrifuged (10000g, 5 minutes, 4 °C), the viscous cell pellet discarded and the supernatant collected and split into two aliquots. The first aliquot was neutralized on ice by adding 0.1 volume of 1 M tricine. This aliquot contained both the oxidized and reduced forms of the pyridine nucleotides, and was also used to determine the protein concentration by a dye-binding assay.42
The other aliquot was heated at 60 °C for 30 minutes in a thermostated water bath to destroy the oxidized forms of the pyridine nucleotides.51 It was then cooled on ice, neutralized by adding 0.1 volume of 1 M tricine, and centrifuged for 10 minutes at 10000g at 4 °C to eliminate any particulate material.
The NADP–NADPH concentration was then determined by using an enzyme cycling assay and standard NADP solutions.52
For cytokine production, a commercial kit (BD Cytometric Bead Array, catalog number 552364 from BD Biosciences) was used.
The cells were then lysed by scraping in 2 ml of lysis buffer (50 mM Hepes (pH 7.5), 4 mM magnesium acetate, 200 mM sorbitol, and 0.1% (w/v) tetradecyldimethylammonio propane sulfonate (SB 3-14)). The lysate was pipetted into a microtube and incubated for 20 minutes on ice to complete cell lysis. For determination of soluble silver, aliquots of the culture media and of the cell lysates were centrifuged for 45 minutes at 16000g, and the upper half of the supernatant was collected.
The samples were mineralized by the addition of one volume of suprapure 65% HNO3 and incubation on a rotating wheel at room temperature for 18 h.
Mineralized samples were diluted in 0.5% (v/v) HNO3 and analysed using an iCAP RQ quadrupole mass instrument (Thermo Fisher Scientific GmbH, Germany) equipped with an ASX-560 auto-sampler (Teledyne CETAC Technologies, Omaha, USA). The instrument was used with a MicroMist U-Series glass concentric nebulizer, a quartz spray chamber cooled at 3 °C, a Qnova quartz torch, a nickel sample cone, and a nickel skimmer cone equipped with a high-sensitivity insert. 24Mg, 25Mg, 107Ag and 109Ag concentrations were determined using standard curves and corrected using an internal standard solution of 103Rh added online. Data integration was done using the Qtegra software (version 2.8.2944.115). The results were normalized using the Mg concentration (4 mM in the cellular extracts and 0.82 mM in culture medium). To take into account the cellular concentration effects, the protein concentration of the extracts was determined by a dye-binding assay.42
The strips were then placed in a Multiphor plate (GE Healthcare), and IEF was carried out with the following electrical parameters: 100 V for 1 hour, then 300 V for 3 hours, then 1000 V for 1 hour, then 3400 V up to 60–70 kVh. After IEF, the gels were equilibrated for 20 minutes in 125 mM Tris, 100 mM HCl, 2.5% SDS, 30% glycerol and 6 M urea.57 They were then transferred on top of the SDS gels and sealed in place with 1% agarose dissolved in 125 mM Tris, 100 mM HCl, 0.4% SDS and 0.005% (w/v) bromophenol blue.
For the global analysis of the spot abundance data, we used directly the spot abundance data as provided by the gel analysis software. The software directly normalizes each spot abundance by the sum of all spot abundances detected on the gel. These relative abundance data were used directly for global analysis using the PAST software suite64 without any transformation. No limitation in the number of principal components was implemented in the principal component analysis.
NanoLC-MS/MS analysis was performed using a nanoACQUITY Ultra-Performance-LC (Waters Corporation, Milford, USA) coupled to a Synapt™ High Definition Mass Spectrometer™ (Waters Corporation, Milford, USA), or to a TripleTOF 5600 (Sciex, Ontario, Canada).
The nanoLC system was composed of an ACQUITY UPLC® CSH130 C18 column (250 mm × 75 μm with a 1.7 μm particle size, Waters Corporation, Milford, USA) and a symmetry C18 precolumn (20 mm × 180 μm with a 5 μm particle size, Waters Corporation, Milford, USA). The solvent system consisted of 0.1% formic acid in water (solvent A) and 0.1% formic acid in acetonitrile (solvent B). 4 μL of the sample were loaded into the enrichment column over 3 min at 5 μL min−1 with 99% of solvent A and 1% of solvent B. Elution of the peptides was performed at a flow rate of 300 nL min−1 with a 8–35% linear gradient of solvent B in 9 minutes.
The Synapt™ High Definition Mass Spectrometer™ (Waters Corporation, Milford, USA) was equipped with a Z-spray ion source and a lock mass system. The system was fully controlled by MassLynx 4.1 SCN639 (Waters Corporation, Milford, USA). The capillary voltage was set at 2.8 kV and the cone voltage at 35 V. Mass calibration of the TOF was achieved using fragment ions from Glu-fibrino-peptide B in the [50;2000] m/z range. Online correction of this calibration was performed with Glu-fibrino-peptide B as the lock-mass. The ion (M + 2H)2+ at m/z 785.8426 was used to calibrate MS data and the fragment ion (M + H)+ at m/z 684.3469 was used to calibrate MS/MS data during the analysis.
For tandem MS experiments, the system was operated with automatic switching between the MS (0.5 s per scan in the m/z range [150;1700]) and MS/MS mode (0.5 s per scan in the m/z range [50;2000]). The two most abundant peptides (intensity threshold 20 counts per s), preferably doubly and triply charged ions, were selected in each MS spectrum for further isolation and CID fragmentation using a collision energy profile. Fragmentation was performed using argon as the collision gas.
Mass data collected during analysis were processed and converted into .pkl files using ProteinLynx Global Server 2.3 (Waters Corporation, Milford, USA). Normal background subtraction was used for both MS and MS/MS with a 5% threshold and polynomial correction of order 5. Smoothing was performed on MS/MS spectra (Savitzky–Golay, 2 iterations, window of 3 channels). Deisotoping was applied for MS (medium deisotoping) and for MS/MS (fast deisotoping).
The TripleTOF 5600 (Sciex, Ontario, Canada) was operated in positive mode, with the following settings: ion spray voltage floating (ISVF) 2300 V, curtain gas (CUR) 10, interface heater temperature (IHT) 150, ion source gas 1 (GS1) 2, declustering potential (DP) 80 V. The information-dependent acquisition (IDA) mode was used with Top 10 MS/MS scans. The MS scan had an accumulation time of 250 ms in the m/z [400;1250] range and the MS/MS scans 100 ms in the m/z [150;1800] range in high sensitivity mode. Switching criteria were set to ions with a charge state of 2–4 and an abundance threshold of more than 500 counts; the exclusion time was set at 4 s. The IDA rolling collision energy script was used for automatically adapting the CE. Mass calibration of the analyser was achieved using peptides from digested BSA. The complete system was fully controlled by AnalystTF 1.7 (Sciex). Raw data collected were processed and converted with MSDataConverter in the .mgf peak list format.
For protein identification, the MS/MS data were interpreted using a local Mascot server with the MASCOT 2.4.1 algorithm (Matrix Science, London, UK) against UniProtKB/SwissProt (version 2016_01, 550299 sequences). The research was carried out in all species. Spectra were searched with a mass tolerance of 15 ppm for MS and 0.05 Da for MS/MS data, allowing a maximum of one trypsin missed cleavage. Carbamidomethylation of cysteine residues and oxidation of methionine residues were specified as variable modifications. Protein identifications were validated with at least two peptides with a Mascot ion score above 30.
Silver accumulation in cells was measured by ICP-MS. The results are summarized in Table 1. They showed a strong accumulation of silver in cells at the end of the 24 hour exposure followed by a moderate loss of total silver. The cells were able to cycle once during the 72 h recovery period, as shown by the increase in the protein amount in the extracts, which resulted in a further reduction of the cellular silver content. A moderate but detectable silver excretion was also evidenced. It should be also noted that the protein concentration is used in toxicology as an indicator for cell number,67,68
Unexposed | Exposed to 20 μg ml−1 AgNPs | 72 h recovery after exposure to AgNPs | |
---|---|---|---|
a This value contains both the soluble silver that is actively excreted by cells during the exposure and the soluble silver that arises from nanoparticle dissolution directly in the culture medium. b Defined as soluble silver in medium/total silver (i.e. silver in medium + silver in cells). | |||
Protein concentration in the cell extract (mg ml−1) | 0.352 ± 0.02 | 0.322 ± 0.02 | 0.657 ± 0.04 |
Total silver concentration in the cell extract (μg l−1) | 0 | 9794 ± 1159 | 8258 ± 1536 |
Soluble silver concentration in the cell extract (μg l−1) | 0 | 39.86 ± 11.83 | 47.57 ± 3.11 |
Soluble silver concentration in culture medium (μg l−1) | 457 ± 64a | 142 ± 4 | |
Normalized silver content (ng Ag mg−1 protein) | 0 | 30![]() |
12![]() |
Soluble silver fraction in cells | 0 | 0.0041 ± 0.0014 | 0.0059 ± 0.001 |
Fraction of silver excretedb | 0 | ND | 0.017 ± 0.003 |
p = 0.031 for the acute vs. control comparison |
p = 0.032 for the recovery vs. control comparison |
p = 0.027 for the acute vs. recovery comparison |
We then divided this dataset of 239 spots into lists (Table S2†). The first list, labelled as “returning spots”, contained the spots for which the amplitude of the change in abundance between the recovery and control stages is lower than the amplitude of the change in abundance between the acute exposure and control stages. 135 spots belonged to this list. It was then divided into subsets. The first subset contained “fast returning spots”. It was defined as spots for which the quantitative change in the acute vs. control comparison shows an amplitude at least twice that in the recover vs. control comparison (i.e. more than 50% convergence). 44 spots belonged to this subset. The other subset of this list contained the 91 “slow returning spots”, for which the return-to-normal trend was lower than 50% in the 72 hour recovery period.
The other list, labelled as “diverging spots”, contained the 104 spots for which the amplitude of the change in abundance between the recovery and control stages is higher than the amplitude of the change in abundance between the acute exposure and control stages. This list was also divided into subsets. The “fast diverging spots” subset was defined as the spots for which the quantitative change in the recover vs. control comparison was at least 1.5 higher than the quantitative change in the acute vs. control comparison (i.e. more than 50% divergence). 53 spots belonged to this subset. The other subset of this list contained the 51 “slow diverging spots”, for which the diverging trend was lower than 50% in the 72 hour recovery period.
Spot number | Protein name | Accession number | Ratio acute/ctrl | T test acute vs. ctrl | Ratio recov/ctrl | T test recov vs. ctrl |
---|---|---|---|---|---|---|
∑: sum of the different spots identified for the same protein. | ||||||
A1 | Casp3 | P70677 | 0.59 | 0.04 | 1.00 | 0.99 |
A2 | Efhd2 | Q9D8Y | 1.24 | 0.02 | 1.18 | 0.23 |
C1 | Actin | P60710 | 1.51 | <0.01 | 1.67 | 0.01 |
C2a | Actn4/1 | P57780 | 1.45 | 0.04 | 1.18 | 0.16 |
C2b | Actn4/2 | P57780 | 1.43 | 0.04 | 1.38 | 0.01 |
C2c | Actn4/3 | P57780 | 1.25 | 0.33 | 1.17 | 0.03 |
C2d | Actn4/4 | P57780 | 1.44 | 0.20 | 1.28 | 0.05 |
C2S | ∑ Actn4 | P57780 | 1.36 | 0.16 | 1.23 | 0.02 |
C3a | Arp2/1 | P61161 | 0.90 | 0.06 | 0.76 | <0.01 |
C3b | Arp2/2 | P61161 | 1.10 | 0.41 | 0.94 | 0.63 |
C3c | Arp2/3 | P61161 | 1.19 | 0.04 | 1.32 | <0.01 |
C3S | ∑ Arp2 | P61161 | 1.15 | 0.07 | 1.14 | 0.06 |
C4a | Arpc2/1 | Q9CVB6 | 0.57 | 0.03 | 0.52 | 0.02 |
C4b | Arpc2/2 | Q9CVB6 | 0.76 | 0.04 | 0.78 | 0.06 |
C4c | Arpc2/3 | Q9CVB6 | 0.91 | 0.51 | 1.12 | 0.41 |
C4S | ∑Arpc2 | Q9CVB6 | 0.80 | 0.11 | 0.90 | 0.39 |
C5a | Arpc5/1 | Q9CPW4 | 0.59 | 0.03 | 0.25 | 0.01 |
C5b | Arpc5/2 | Q9CPW4 | 1.06 | 0.51 | 0.99 | 0.90 |
C5S | ∑ Arpc5 | Q9CPW4 | 0.88 | 0.30 | 0.71 | 0.04 |
C6a | Capg/1 | P24452 | 0.81 | 0.01 | 1.00 | 0.97 |
C6b | Capg/2 | P24452 | 1.16 | 0.15 | 1.04 | 0.58 |
C6S | ∑ capg | P24452 | 0.35 | 0.39 | 0.35 | 0.39 |
C7 | Capza2 | P47754 | 1.30 | 0.01 | 1.28 | 0.01 |
C8a | Capzb/1 | P47757 | 1.07 | 0.37 | 1.20 | 0.06 |
C8b | Capzb/2 | P47757 | 0.80 | 0.02 | 1.01 | 0.95 |
C8S | ∑ capzb | P47757 | 0.98 | 0.60 | 1.13 | 0.14 |
C9a | Cof/1 | P18760 | 0.67 | 0.05 | 0.60 | 0.03 |
C9b | Cof/2 | P18760 | 1.49 | 0.01 | 1.26 | 0.10 |
C9c | Cof/3 | P18760 | 0.74 | <0.01 | 0.73 | <0.01 |
C9S | ∑ Cof | P18760 | 0.86 | 0.02 | 0.79 | <0.01 |
C10a | Dyncli2/1 | O88487 | 1.21 | 0.49 | 2.20 | <0.01 |
C10b | Dyncli2/2 | O88487 | 1.43 | 0.26 | 1.69 | 0.04 |
C10S | ∑ dyncli2 | O88487 | 1.36 | 0.30 | 1.84 | 0.01 |
C11a | Gelsolin/1 | P13020 | 1.16 | 0.44 | 0.94 | 0.71 |
C11b | Gelsolin/2 | P13020 | 1.16 | 0.36 | 1.00 | 0.97 |
C11c | Gelsolin/3 | P13020 | 1.39 | 0.16 | 1.27 | 0.26 |
C11d | Gelsolin/4 | P13020 | 1.57 | 0.05 | 1.32 | 0.13 |
C11S | ∑ gelsolin | P13020 | 1.33 | 0.15 | 1.15 | 0.39 |
C12 | Gmfg | Q9ERL7 | 0.54 | <0.01 | 0.66 | <0.01 |
C13 | Lasp1 | Q61792 | 0.41 | <0.01 | 0.38 | <0.01 |
C14 | Ml12b | Q3THE2 | 0.92 | 0.51 | 0.81 | 0.01 |
C15a | Moesin/1 | P26041 | 0.94 | 0.75 | 0.86 | 0.41 |
C15b | Moesin/2 | P26041 | 0.90 | 0.53 | 0.78 | 0.08 |
C15c | Moesin/3 | P26041 | 1.22 | 0.17 | 0.89 | 0.13 |
C15d | Moesin/4 | P26041 | 1.18 | 0.07 | 1.09 | 0.10 |
C15e | Moesin/5 | P26041 | 1.52 | 0.03 | 1.24 | 0.04 |
C15S | ∑ Moesin | P26041 | 1.20 | 0.08 | 1.01 | 0.91 |
C16 | Mtap | Q9CQ65 | 1.04 | 0.83 | 1.30 | 0.02 |
C17 | RhoA | Q9QUI0 | 0.59 | <0.01 | 0.76 | 0.02 |
C18a | Rhogdi1/1 | Q99PT1 | 0.71 | <0.01 | 0.67 | <0.01 |
C18b | Rhogdi1/2 | Q99PT1 | 1.16 | 0.13 | 1.11 | 0.08 |
C18S | ∑ RhoGdi1 | Q99PT1 | 0.99 | 0.85 | 0.95 | 0.28 |
C19a | Rhogdi2/1 | Q61599 | 0.64 | <0.01 | 0.54 | <0.01 |
C19b | Rhogdi2/2 | Q61599 | 1.00 | 0.99 | 0.95 | 0.17 |
C19S | ∑ rhogdi2 | Q61599 | 0.87 | 0.02 | 0.80 | <0.01 |
C20a | Stmn/1 | P54227 | 0.84 | 0.19 | 0.70 | 0.01 |
C20b | Stmn/2 | P54227 | 0.60 | 0.02 | 0.53 | 0.01 |
C20c | Stmn/3 | P54227 | 0.88 | 0.14 | 0.76 | 0.01 |
C20S | ∑ Stmn | P54227 | 0.83 | 0.05 | 0.71 | 0.01 |
C21a | Sw70/1 | Q6A028 | 1.63 | 0.02 | 1.50 | 0.20 |
C21b | Sw70/2 | Q6A028 | 2.02 | 0.02 | 1.76 | 0.08 |
C21S | ∑ Sw70 | Q6A028 | 1.77 | 0.01 | 1.60 | 0.14 |
C22 | Tbcb | Q9D1E6 | 1.31 | 0.03 | 1.11 | 0.45 |
C23a | Tctp/1 | P63028 | 0.60 | <0.01 | 0.54 | <0.01 |
C23b | Tctp/2 | P63028 | 0.84 | 0.03 | 0.85 | 0.05 |
C23S | ∑ Tctp | P63028 | 0.84 | 0.03 | 0.72 | <0.01 |
C24a | Twf1/1 | Q91YR1 | 0.58 | <0.01 | 0.93 | 0.39 |
C24b | Twf1/2 | Q91YR1 | 0.25 | 0.01 | 0.86 | 0.52 |
C24S | ∑ Twf1 | Q91YR1 | 0.40 | <0.01 | 0.89 | 0.47 |
C25 | Twf2 | Q9Z0P5 | 0.70 | 0.01 | 0.60 | 0.01 |
C26 | Vime | P20152 | 1.54 | 0.05 | 1.35 | 0.04 |
C27a | Vinculin/1 | Q64727 | 1.28 | 0.25 | 1.24 | 0.11 |
C27b | Vinculin/2 | Q64727 | 1.32 | 0.07 | 1.16 | 0.24 |
C27c | Vinculin/3 | Q64727 | 1.63 | 0.01 | 1.12 | 0.28 |
C27S | ∑ Vinculin | Q64727 | 1.39 | 0.07 | 1.18 | 0.16 |
D1 | Cbx1 | P83917 | 0.91 | 0.27 | 0.72 | <0.01 |
D2a | Ddb1/1 | Q3U1J4 | 1.74 | 0.04 | 1.40 | 0.16 |
D2b | Ddb1/2 | Q3U1J4 | 0.93 | 0.65 | 0.78 | 0.21 |
D2S | ∑ Ddb1 | Q3U1J4 | 1.31 | 0.14 | 1.07 | 0.73 |
D3 | Nt5c | Q9JM14 | 1.18 | 0.08 | 1.39 | 0.03 |
D4a | Pcna/1 | P17918 | 0.57 | <0.01 | 0.57 | <0.01 |
D4b | Pcna/2 | P17918 | 0.82 | 0.02 | 0.80 | 0.03 |
D4c | Pcna/3 | P17918 | 1.00 | 0.92 | 0.98 | 0.73 |
D4S | ∑ Pcna | P17918 | 0.84 | <0.01 | 0.82 | 0.02 |
D5 | Ruvb2 | Q9WTM5 | 1.46 | 0.06 | 1.42 | 0.04 |
E1a | 6pgd/1 | Q9DCD0 | 0.88 | 0.05 | 0.95 | 0.42 |
E1b | 6pgd/2 | Q9DCD0 | 1.05 | 0.32 | 1.01 | 0.90 |
E1S | ∑ 6pgd | Q9DCD0 | 0.98 | 0.45 | 0.99 | 0.82 |
E2a | Aacs/1 | Q9D2R0 | 1.79 | 0.11 | 1.39 | 0.52 |
E2b | Aacs/2 | Q9D2R0 | 3.12 | 0.01 | 2.56 | <0.01 |
E2S | ∑ Aacs | Q9D2R0 | 2.17 | 0.01 | 1.73 | 0.16 |
E3 | Acadl | P51174 | 1.13 | 0.14 | 1.27 | 0.01 |
E4a | Eno1a/1 | P17182 | 0.99 | 0.96 | 0.89 | 0.28 |
E4b | Eno1a/2 | P17182 | 1.10 | 0.50 | 0.89 | 0.42 |
E4c | Eno1a/3 | P17182 | 1.18 | 0.10 | 1.21 | 0.10 |
E4d | Eno1a/4 | P17182 | 1.50 | <0.01 | 1.42 | <0.01 |
E4S | ∑ Eno1A | P17182 | 1.26 | 0.02 | 1.19 | 0.06 |
E5 | GalK | Q9R0N0 | 1.45 | <0.01 | 1.50 | 0.03 |
E6 | Gapdh | P16858 | 0.56 | <0.01 | 0.60 | <0.01 |
E7 | Gpd1L | Q3ULJ0 | 2.27 | <0.01 | 1.42 | 0.14 |
E8a | Hxk3/1 | Q3TRM8 | 1.91 | 0.02 | 1.80 | 0.03 |
E8b | Hxk3/2 | Q3TRM8 | 1.23 | 0.04 | 1.22 | 0.09 |
E8c | Hxk3/3 | Q3TRM8 | 1.52 | 0.02 | 1.52 | 0.02 |
E8S | ∑ Hxk3 | Q3TRM8 | 1.51 | 0.01 | 1.49 | 0.01 |
E9 | Idhc | O88844 | 1.24 | 0.02 | 1.19 | <0.01 |
E10a | Ldha/1 | P06151 | 0.79 | 0.02 | 0.86 | 0.06 |
E10b | Ldha/2 | P06151 | 0.97 | 0.81 | 0.89 | 0.40 |
E10S | ∑ Ldha | P06151 | 0.89 | 0.26 | 0.88 | 0.21 |
E11a | Mdhc/1 | P14152 | 0.47 | 0.02 | 0.72 | 0.11 |
E11b | Mdhc/2 | P14152 | 0.83 | 0.01 | 0.84 | 0.01 |
E11c | Mdhc/3 | P14152 | 1.38 | 0.02 | 1.40 | <0.01 |
E11S | ∑ Mdhc | P14152 | 1.01 | 0.91 | 1.05 | 0.20 |
E12 | Pckgm | Q8BH04 | 1.30 | 0.20 | 0.61 | 0.01 |
E13 | Pfkap | Q9WUA3 | 0.84 | 0.01 | 0.86 | 0.01 |
E14 | Pfkl | P12382 | 1.83 | 0.01 | 1.62 | 0.03 |
E15a | Pgls/1 | Q9CQ60 | 0.78 | 0.02 | 0.97 | 0.75 |
E15b | Pgls/2 | Q9CQ60 | 1.11 | 0.16 | 1.22 | 0.01 |
E16 | Pgp | Q8CHP8 | 1.37 | 0.02 | 1.60 | 0.05 |
E17a | Taldo1 | Q93092 | 0.80 | 0.02 | 0.66 | <0.01 |
E17b | Taldo/2 | Q93092 | 1.17 | 0.12 | 1.18 | 0.08 |
E17c | Taldo/3 | Q93092 | 1.24 | 0.02 | 1.23 | <0.01 |
E17d | Taldo/4 | Q93092 | 0.81 | 0.06 | 0.79 | 0.02 |
E17e | Taldo/5 | Q93092 | 1.22 | 0.06 | 1.36 | 0.02 |
E17S | ∑ Taldo | Q93092 | 1.08 | 0.16 | 1.09 | 0.06 |
E18a | Tpis/1 | P17751 | 0.74 | 0.01 | 0.82 | 0.04 |
E18b | Tpis/2 | P17751 | 1.04 | 0.61 | 1.20 | 0.09 |
E18c | Tpis/3 | P17751 | 1.45 | 0.04 | 1.43 | 0.05 |
E18S | ∑ Tpis | P17751 | 1.09 | 0.29 | 1.16 | 0.17 |
F1 | Cdk4 | P30285 | 0.69 | <0.01 | 0.71 | <0.01 |
F2 | Cdk6 | Q64261 | 0.71 | 0.04 | 0.77 | 0.02 |
F3a | Ndrg1/1 | Q62433 | 1.32 | 0.09 | 1.52 | 0.04 |
F3b | Ndrg1/2 | Q62433 | 1.38 | 0.01 | 1.33 | 0.02 |
F3S | ∑ ndrg1 | Q62433 | 1.34 | 0.02 | 1.44 | 0.03 |
F4 | Pa2 g4 | P50580 | 1.50 | <0.01 | 1.31 | 0.01 |
F5a | Vma5a/1 | Q99KC8 | 1.60 | 0.06 | 0.99 | 0.97 |
F5b | Vma5a/2 | Q99KC8 | 1.31 | 0.11 | 1.19 | 0.10 |
F5c | Vma5a/3 | Q99KC8 | 1.84 | <0.01 | 1.59 | 0.02 |
F5S | ∑ Vma5a | Q99KC8 | 1.54 | 0.02 | 1.25 | 0.12 |
G1 | GalE | Q8R059 | 0.50 | 0.03 | 1.09 | 0.50 |
G2 | Gnpda1 | O88958 | 0.47 | <0.01 | 0.63 | 0.01 |
G3 | Gt25c | Q8K297 | 1.53 | 0.01 | 1.48 | 0.01 |
G4 | Mlec | Q6ZQI3 | 1.49 | 0.02 | 1.79 | <0.01 |
G5a | Naga/1 | Q8JZV7 | 1.19 | 0.07 | 1.19 | 0.06 |
G5b | Naga/2 | Q8JZV7 | 1.05 | 0.56 | 1.18 | 0.08 |
G5S | ∑ Naga | Q8JZV7 | 1.12 | 0.16 | 1.19 | 0.06 |
H1 | Adh5 | P28474 | 0.72 | 0.09 | 0.74 | 0.11 |
H2a | Aldr/1 | P45376 | 0.57 | 0.01 | 0.66 | 0.01 |
H2b | Aldr/2 | P45376 | 0.84 | 0.09 | 0.73 | 0.04 |
H2c | Aldr/3 | P45376 | 1.18 | 0.04 | 1.25 | 0.01 |
H2S | ∑ Aldr | P45376 | 0.85 | 0.02 | 0.89 | 0.10 |
H3 | Aldr2 | P47738 | 1.16 | 0.08 | 1.23 | <0.01 |
H4a | Bvra/1 | Q9CY64 | 0.54 | 0.02 | 0.62 | 0.03 |
H4b | Bvra/2 | Q9CY64 | 1.23 | 0.08 | 1.30 | 0.06 |
H4S | ∑ Bvra | Q9CY64 | 0.98 | 0.81 | 1.06 | 0.54 |
H5 | Bvrb | Q923D2 | 1.45 | 0.01 | 0.96 | 0.77 |
H6 | Ca13 | Q9D6N1 | 0.62 | 0.01 | 0.83 | 0.11 |
H7a | Esd/1 | Q9R0P3 | 0.56 | <0.01 | 0.64 | 0.01 |
H7b | Esd/2 | Q9R0P3 | 0.78 | <0.01 | 0.92 | 0.15 |
H7S | ∑ Esd | Q9R0P3 | 0.73 | <0.01 | 0.86 | 0.02 |
H8 | Frih | P09528 | 0.61 | 0.01 | 0.67 | 0.02 |
H9 | Gclm | O09172 | 1.32 | 0.01 | 0.98 | 0.86 |
H10 | Hmox2 | O70252 | 1.53 | <0.01 | 1.03 | 0.69 |
H11a | Lgul/1 | Q9CPU0 | 0.80 | 0.04 | 0.77 | 0.05 |
H11b | Lgul/2 | Q9CPU0 | 1.10 | 0.24 | 1.10 | 0.16 |
H11S | ∑ Lgul | Q9CPU0 | 1.00 | 0.99 | 1.00 | 0.96 |
L1 | Anxa1 | P10107 | 0.78 | 0.02 | 1.11 | 0.06 |
L2a | Anxa2/1 | P07356 | 0.79 | 0.01 | 0.60 | <0.01 |
L2b | Anxa2/2 | P07356 | 0.44 | <0.01 | 0.56 | <0.01 |
L2S | ∑ Anxa2 | P07356 | 0.67 | <0.01 | 0.59 | <0.01 |
L3 | Anxa3 | O35639 | 1.34 | 0.01 | 1.30 | 0.02 |
L4 | Anxa4 | P97429 | 1.30 | 0.01 | 1.33 | <0.01 |
L5a | Anxa5/1 | P48036 | 0.88 | 0.02 | 0.81 | <0.01 |
L5b | Anxa5/2 | P48036 | 1.09 | 0.34 | 1.07 | 0.37 |
L5S | ∑ Anxa5 | P48036 | 0.95 | 0.35 | 0.90 | 0.08 |
L6 | Anxa6 | P14824 | 1.54 | 0.02 | 1.80 | <0.01 |
L7 | Anxa7 | Q07076 | 1.14 | 0.44 | 1.57 | 0.02 |
L8 | Idi1 | P58044 | 1.40 | 0.02 | 1.34 | 0.01 |
L9 | Lypla2 | Q9WTL7 | 1.07 | 0.28 | 1.22 | 0.02 |
L10 | Mbd3 | Q9Z2D8 | 1.52 | 0.02 | 1.77 | <0.01 |
L11 | Pipna | P53810 | 1.22 | 0.11 | 1.32 | 0.01 |
L12 | Ppt1 | O88531 | 1.24 | 0.14 | 1.34 | 0.03 |
M1a | Clic4/1 | Q9QYB1 | 0.90 | 0.12 | 0.95 | 0.58 |
M1b | Clic4/2 | Q9QYB1 | 1.27 | 0.02 | 1.27 | 0.04 |
M1S | ∑ Clic4 | Q9QYB1 | 1.12 | 0.04 | 1.14 | 0.01 |
M2 | Clybl | Q8R4N0 | 1.80 | 0.03 | 1.41 | 0.19 |
M3 | Gatm | Q9D964 | 0.68 | 0.01 | 0.54 | 0.01 |
M4 | Hmgcl | P38060 | 0.61 | <0.01 | 0.76 | 0.07 |
M5 | Nduv2 | Q9D6J6 | 1.24 | 0.01 | 1.49 | 0.05 |
M6 | Oat | P29758 | 1.30 | 0.05 | 1.50 | 0.01 |
M7 | Odba | P50136 | 1.54 | 0.02 | 1.28 | 0.12 |
M8 | Phb | P67778 | 1.12 | 0.16 | 1.18 | 0.05 |
M9 | Tmem11 | Q8BK08 | 1.78 | 0.04 | 1.46 | 0.41 |
M10 | Vdac2 | Q60930 | 0.63 | 0.02 | 0.72 | 0.05 |
M11a | Atpb ac | P56480 | 1.23 | 0.16 | 1.41 | 0.03 |
M11b | Atpb bas | P56480 | 1.60 | 0.04 | 1.58 | <0.01 |
M11S | ∑Atpb | P56480 | 1.41 | 0.06 | 1.49 | <0.01 |
M12 | Clpp | O88696 | 1.28 | 0.33 | 1.84 | 0.02 |
M13a | Trap1/1 | Q9CQN1 | 1.74 | 0.02 | 1.32 | 0.13 |
M13b | Trap1/2 | Q9CQN1 | 1.21 | 0.25 | 0.92 | 0.64 |
M13S | ∑Trap1 | Q9CQN1 | 1.47 | 0.03 | 1.11 | 0.51 |
N1a | Aprt/1 | P08030 | 0.52 | <0.01 | 0.72 | <0.01 |
N1b | Aprt/2 | P08030 | 1.06 | 0.43 | 1.08 | 0.39 |
N1S | ∑ Aprt | P08030 | 0.92 | 0.19 | 0.99 | 0.85 |
N2 | Bpnt1 | Q9Z0S1 | 1.77 | 0.02 | 1.87 | 0.01 |
N3 | Guaa | Q3THK7 | 1.42 | 0.02 | 1.12 | 0.25 |
N4 | Hint1 | P70349 | 1.46 | 0.30 | 0.64 | 0.02 |
N5 | Ndka | P15532 | 0.42 | 0.03 | 0.75 | 0.22 |
N6 | Paps1 | Q60967 | 5.54 | 0.04 | 2.44 | 0.01 |
N7a | Pnph/1 | P23492 | 0.83 | 0.12 | 0.72 | 0.05 |
N7b | Pnph/2 | P23492 | 1.25 | 0.02 | 1.22 | 0.08 |
N7S | ∑ Pnph | P23492 | 1.08 | 0.17 | 1.02 | 0.76 |
N8a | Prps1/1 | Q9D7G0 | 0.69 | 0.01 | 0.92 | 0.48 |
N8b | Prps1/2 | Q9D7G0 | 0.57 | <0.01 | 0.77 | 0.16 |
N8c | Prps1/3 | Q9D7G0 | 0.52 | 0.01 | 0.83 | 0.21 |
N8S | ∑ Prps1 | Q9D7G0 | 0.60 | <0.01 | 0.83 | 0.14 |
N9a | Pur4/1 | Q5SUR0 | 1.71 | 0.14 | 1.27 | 0.07 |
N9b | Pur4/2 | Q5SUR0 | 2.53 | 0.01 | 2.01 | 0.01 |
N9c | Pur4/3 | Q5SUR0 | 1.15 | 0.53 | 1.25 | 0.22 |
N9S | ∑ Pur4 | Q5SUR0 | 1.65 | 0.07 | 1.43 | 0.01 |
N10 | Pur9 | Q9CWJ9 | 1.16 | 0.27 | 1.23 | 0.01 |
N11 | Nt5c3b | Q3UFY7 | 1.55 | <0.01 | 1.64 | 0.01 |
O1 | Prx1ox | P35700 | 0.58 | 0.02 | 0.70 | 0.04 |
O2a | Prx3/1 | P20108 | 0.93 | 0.69 | 1.13 | 0.08 |
O2b | Prx3/2 | P20108 | 1.23 | 0.06 | 1.32 | 0.01 |
O2S | ∑ Prx3 | P20108 | 1.16 | 0.22 | 1.27 | 0.01 |
O3 | Prx4 | O08807 | 0.75 | 0.06 | 0.56 | 0.01 |
O4a | Prx6/1 | O08709 | 0.56 | 0.02 | 0.45 | 0.01 |
O4b | Prx6/2 | O08709 | 1.05 | 0.64 | 0.97 | 0.76 |
O4S | ∑ Prx6 | O08709 | 0.96 | 0.63 | 0.87 | 0.23 |
P1 | Aars | Q8BGQ7 | 1.65 | 0.01 | 1.18 | 0.20 |
P2a | Eef2/1 | P58252 | 0.77 | 0.02 | 0.63 | <0.01 |
P2b | Eef2/2 | P58252 | 1.06 | 0.41 | 0.96 | 0.39 |
P2c | Eef2/3 | P58252 | 0.84 | 0.07 | 0.90 | 0.20 |
P2d | Eef2/4 | P58252 | 0.99 | 0.88 | 0.98 | 0.61 |
P2e | Eef2/5 | P58252 | 1.39 | 0.01 | 1.29 | 0.01 |
P2S | ∑ Eef2 | P58252 | 1.02 | 0.72 | 0.97 | 0.43 |
P3 | If3g | Q9Z1D1 | 0.77 | 0.31 | 0.56 | 0.03 |
P4a | If5a/1 | P63242 | 0.54 | <0.01 | 0.62 | <0.01 |
P4d | If5a/2 | P63242 | 1.07 | 0.26 | 1.03 | 0.58 |
P4S | ∑ If5a | P63242 | 0.86 | 0.04 | 0.87 | 0.04 |
P5 | If4a1 | P60843 | 1.55 | 0.01 | 1.51 | 0.01 |
P6 | If4a3 | Q91VC3 | 1.27 | 0.05 | 1.09 | 0.28 |
P7 | Rla0 | P14869 | 1.28 | 0.07 | 1.33 | 0.04 |
P8 | Rs4y1 | P62702 | 0.50 | 0.04 | 0.62 | 0.07 |
P9a | Sars/1 | P26638 | 1.42 | 0.03 | 1.05 | 0.59 |
P9b | Sars/2 | P26638 | 1.52 | 0.04 | 1.10 | 0.42 |
P9S | ∑ Sars | P26638 | 1.48 | 0.02 | 1.08 | 0.46 |
Q1a | Dpp3/1 | Q99KK7 | 1.28 | 0.22 | 1.75 | <0.01 |
Q1b | Dpp3/2 | Q99KK7 | 1.55 | 0.04 | 1.84 | <0.01 |
Q1S | ∑ Dpp3 | Q99KK7 | 1.40 | 0.09 | 1.79 | <0.01 |
Q2a | Hsp74/1 | Q61316 | 0.77 | 0.22 | 0.77 | 0.23 |
Q2b | Hsp74/2 | Q61316 | 1.33 | 0.08 | 1.18 | 0.23 |
Q2c | Hsp74/3 | Q61316 | 1.31 | 0.09 | 1.14 | 0.29 |
Q2d | Hsp74/4 | Q61316 | 1.26 | 0.15 | 1.31 | <0.01 |
Q2S | ∑ Hsp74 | Q61316 | 1.07 | 0.58 | 1.03 | 0.80 |
Q3a | Hyou1/1 | Q9JKR6 | 1.18 | 0.25 | 0.83 | 0.20 |
Q3b | Hyou1/2 | Q9JKR6 | 1.38 | 0.01 | 1.18 | 0.03 |
Q3c | Hyou1/3 | Q9JKR6 | 1.35 | 0.01 | 1.21 | 0.05 |
Q3d | Hyou1/4 | Q9JKR6 | 0.80 | 0.23 | 0.79 | 0.11 |
Q3S | ∑ Hyou1 | Q9JKR6 | 1.04 | 0.67 | 0.93 | 0.27 |
Q4 | Lxn | P70202 | 0.98 | 0.80 | 1.26 | <0.01 |
Q5 | Pfd2 | O70591 | 0.72 | 0.01 | 0.70 | 0.01 |
Q6a | Ppce/1 | Q9QUR6 | 1.40 | 0.15 | 1.78 | 0.01 |
Q6b | Ppce/2 | Q9QUR6 | 1.58 | 0.04 | 1.66 | 0.01 |
Q6S | ∑ Ppce | Q9QUR6 | 1.50 | 0.07 | 1.71 | 0.01 |
Q7a | Ppia/1 | P17742 | 0.68 | 0.11 | 0.57 | 0.05 |
Q7b | Ppia/2 | P17742 | 0.80 | 0.21 | 0.79 | 0.21 |
Q7c | Ppia/3 | P17742 | 0.81 | 0.09 | 0.99 | 0.97 |
Q7S | ∑ Ppia | P17742 | 0.78 | 0.09 | 0.83 | 0.21 |
Q8a | Spb6/1 | Q60854 | 1.66 | 0.02 | 1.49 | 0.01 |
Q8b | Spb6/2 | Q60854 | 1.43 | 0.01 | 1.42 | 0.01 |
Q8S | ∑ Spb6 | Q60854 | 1.57 | 0.01 | 1.46 | <0.01 |
Q9 | Stip1 | Q60864 | 1.72 | <0.01 | 0.94 | 0.67 |
Q10 | Tcpa | P11983 | 1.55 | 0.02 | 1.09 | 0.55 |
R1a | Btf3/1 | Q64152 | 0.56 | 0.01 | 0.50 | 0.01 |
R1b | Btf3/2 | Q64152 | 0.76 | 0.05 | 0.76 | 0.05 |
R1S | ∑ Btf3 | Q64152 | 0.66 | 0.02 | 0.64 | 0.01 |
R2 | Dcps | Q9DAR7 | 1.30 | <0.01 | 1.20 | 0.02 |
R3 | Ddx39a | Q8VDW0 | 1.64 | 0.03 | 1.31 | 0.02 |
R4 | Ddx39b | Q9Z1N5 | 1.59 | 0.03 | 1.35 | 0.11 |
R5 | Mgn | P61327 | 0.96 | 0.60 | 0.56 | 0.01 |
R6a | Sf3b2/1 | Q3UJB0 | 1.03 | 0.91 | 0.87 | 0.49 |
R6b | Sf3b2/2 | Q3UJB0 | 1.16 | 0.44 | 1.32 | <0.01 |
R6c | Sf3b2/3 | Q3UJB0 | 1.26 | 0.38 | 1.80 | <0.01 |
R6S | ∑ Sf3b2 | Q3UJB0 | 1.13 | 0.54 | 1.27 | 0.03 |
R7 | ExoS4 | Q921I9 | 0.88 | 0.42 | 0.51 | 0.02 |
R8 | ExoS6 | Q8BTW3 | 1.50 | 0.02 | 1.53 | 0.01 |
S1a | 14-3-3 gam/1 | P61982 | 0.85 | 0.28 | 0.67 | 0.02 |
S1b | 14-3-3 gam/2 | P61982 | 0.83 | 0.06 | 0.91 | 0.24 |
S1S | ∑ 14-3-3 gam | P61982 | 0.84 | 0.10 | 0.82 | 0.07 |
S2 | 14-3-3 th | P68254 | 1.31 | 0.01 | 1.19 | 0.04 |
S3 | Cab39 | Q06138 | 0.70 | 0.03 | 0.62 | <0.01 |
S4 | Fam49b | Q921M7 | 1.47 | 0.02 | 1.59 | 0.05 |
S5 | Gbb1 | P62874 | 1.17 | 0.03 | 1.18 | 0.03 |
S6a | Gnai2/1 | P08752 | 1.33 | 0.06 | 1.42 | 0.05 |
S6b | Gnai2/2 | P08752 | 1.51 | <0.01 | 1.67 | 0.01 |
S6S | ∑ Gnai2 | P08752 | 1.45 | 0.01 | 1.59 | 0.02 |
S7 | Gnb2L1 | P68040 | 0.89 | 0.02 | 0.81 | <0.01 |
S8 | Grb2 | Q60631 | 1.06 | 0.83 | 1.46 | 0.02 |
S9 | Igbp1 | Q61249 | 1.36 | 0.06 | 1.36 | 0.04 |
S10 | In35 | Q9D8C4 | 1.25 | 0.04 | 1.21 | 0.14 |
S11 | Inpp | P49442 | 2.46 | 0.01 | 1.76 | 0.17 |
S12 | Ppp1ca | P62137 | 1.17 | 0.07 | 1.27 | 0.02 |
S13a | Ppp1r7/1 | Q3UM45 | 0.30 | 0.02 | 0.38 | 0.04 |
S13b | Ppp1r7/2 | Q3UM45 | 0.67 | 0.02 | 0.73 | 0.08 |
S13c | Ppp1r7/3 | Q3UM45 | 0.90 | 0.51 | 0.94 | 0.72 |
S13S | ∑ Ppp1r7 | Q3UM45 | 0.61 | 0.03 | 0.68 | 0.06 |
S14 | Ppp6 | O00743 | 1.48 | 0.03 | 1.04 | 0.82 |
S15a | Snd1/1 | Q78PY7 | 0.65 | 0.03 | 0.66 | 0.04 |
S15b | Snd1/2 | Q78PY7 | 0.59 | 0.02 | 0.50 | 0.01 |
S15c | Snd1/3 | Q78PY7 | 0.89 | 0.34 | 0.78 | 0.18 |
S15d | Snd1/4 | Q78PY7 | 1.11 | 0.34 | 0.91 | 0.47 |
S15S | ∑ Snd1 | Q78PY7 | 0.79 | <0.01 | 0.70 | 0.01 |
U1 | Chip | Q9WUD1 | 0.59 | <0.01 | 0.73 | 0.11 |
U2 | Csn4 | O88544 | 1.34 | 0.03 | 1.37 | 0.03 |
U3a | Prs8/1 | P62196 | 0.60 | <0.01 | 0.65 | <0.01 |
U3b | Prs8/2 | P62196 | 0.85 | 0.04 | 0.85 | 0.22 |
U3S | ∑ Prs8 | P62196 | 0.31 | 0.33 | 0.32 | 0.34 |
U4a | Psa2/1 | P49722 | 0.58 | <0.01 | 0.58 | <0.01 |
U4b | Psa2/2 | P49722 | 1.15 | 0.12 | 1.28 | 0.07 |
U4S | ∑ Psa2 | P49722 | 0.86 | 0.04 | 0.92 | 0.36 |
U5a | Psa5/1 | Q9Z2U1 | 0.80 | 0.01 | 0.84 | 0.11 |
U5b | Psa5/2 | Q9Z2U1 | 0.92 | 0.33 | 0.99 | 0.83 |
U5S | ∑ Psa5 | Q9Z2U1 | 0.86 | 0.07 | 0.92 | 0.22 |
U6 | Psb10 | O35955 | 0.73 | 0.12 | 0.43 | 0.02 |
U7a | Psb2/1 | Q9R1P3 | 0.67 | 0.02 | 0.76 | 0.14 |
U7b | Psb2/2 | Q9R1P3 | 0.90 | 0.36 | 1.01 | 0.91 |
U7S | ∑ Psb2 | Q9R1P3 | 0.82 | 0.09 | 0.92 | 0.50 |
U8 | Psb3 mod | Q9R1P1 | 0.47 | 0.01 | 0.65 | <0.01 |
U9a | Psb4/1 | P99026 | 0.74 | 0.05 | 0.69 | 0.03 |
U9b | Psb4/2 | P99026 | 0.98 | 0.71 | 0.94 | 0.25 |
U9S | ∑ Psb4 | P99026 | 0.90 | 0.05 | 0.86 | 0.01 |
U10 | Psmd14 | O35593 | 1.03 | 0.61 | 1.32 | <0.01 |
U11a | Psmd2/1 | Q8VDM4 | 0.82 | 0.22 | 1.12 | 0.24 |
U11b | Psmd2/2 | Q8VDM4 | 1.10 | 0.11 | 1.09 | 0.02 |
U11c | Psmd2/3 | Q8VDM4 | 1.39 | 0.02 | 1.13 | 0.17 |
U11S | ∑ Psmd2 | Q8VDM4 | 1.03 | 0.70 | 1.12 | 0.12 |
U12 | Psmd7 | P26516 | 0.68 | 0.03 | 0.68 | 0.02 |
U13 | Psme1 | P97371 | 1.40 | <0.01 | 1.40 | <0.01 |
U14a | Psme2/1 | P97372 | 1.00 | 0.98 | 0.71 | <0.01 |
U14b | Psme2/2 | P97372 | 1.22 | <0.01 | 1.07 | 0.34 |
U14S | ∑ Psme2 | P97372 | 1.15 | <0.01 | 0.96 | 0.50 |
U15 | Ubl7 | Q91W67 | 0.41 | 0.02 | 0.88 | 0.55 |
V1 | Asna1 | O54984 | 1.29 | 0.09 | 1.46 | <0.01 |
V2 | Chm2a | Q9DB34 | 0.83 | <0.01 | 0.92 | 0.09 |
V3 | CopE | O89079 | 1.35 | 0.01 | 1.41 | 0.07 |
V4 | Emc2 | Q9CRD2 | 0.56 | 0.01 | 0.84 | 0.07 |
V5 | Erp29 | P57759 | 0.78 | 0.12 | 0.68 | 0.05 |
V6 | Mss4 | Q91X96 | 0.52 | 0.04 | 0.64 | 0.10 |
V7 | Nsf1c | Q9CZ44 | 1.55 | 0.01 | 1.51 | 0.01 |
V8 | Pef1 | Q8BFY6 | 0.64 | 0.05 | 0.50 | 0.02 |
V9 | Snaa | Q9DB05 | 1.53 | <0.01 | 1.99 | 0.05 |
V10 | Snap23 | O09044 | 0.53 | 0.02 | 0.34 | 0.01 |
V11 | Stxb2 | Q64324 | 2.07 | <0.01 | 1.30 | 0.09 |
V12a | Tera/1 | Q01853 | 1.31 | 0.13 | 1.18 | 0.16 |
V12b | Tera/2 | Q01853 | 1.49 | 0.02 | 1.06 | 0.38 |
V12c | Tera/3 | Q01853 | 1.39 | 0.02 | 1.29 | 0.04 |
V12d | Tera/4 | Q01853 | 1.48 | 0.10 | 1.35 | <0.01 |
V12S | ∑ Tera | Q01853 | 1.43 | 0.02 | 1.25 | 0.01 |
V13 | Tpd52 | Q62393 | 1.42 | 0.04 | 1.31 | 0.10 |
V14 | Vat1 | Q62465 | 1.48 | 0.01 | 1.22 | 0.15 |
V15 | Vps29 | Q9QZ88 | 1.30 | 0.05 | 1.29 | 0.08 |
V16a | Vps35/1 | Q9EQH3 | 2.44 | 0.16 | 2.33 | <0.01 |
V16b | Vps35/2 | Q9EQH3 | 1.80 | 0.17 | 2.03 | 0.01 |
V16c | Vps35/3 | Q9EQH3 | 1.53 | 0.33 | 2.25 | <0.01 |
V16S | ∑ Vps35 | Q9EQH3 | 1.79 | 0.18 | 2.20 | <0.01 |
V17 | Vta1 | Q9CR26 | 2.84 | 0.01 | 2.97 | 0.02 |
X1 | Pdxk | Q8K183 | 1.29 | <0.01 | 1.45 | 0.02 |
X2 | Spee | Q64674 | 1.55 | 0.02 | 1.43 | 0.07 |
X3 | Sps1 | Q8BH69 | 1.49 | 0.02 | 1.95 | 0.01 |
X4a | Ran/1 | P62827| | 0.83 | 0.04 | 0.87 | 0.12 |
X4b | Ran/2 | P62827| | 1.18 | 0.08 | 1.11 | 0.17 |
X4S | ∑ Ran | P62827| | 1.03 | 0.62 | 1.00 | 0.98 |
Y1 | CatD | P18242 | 1.38 | 0.01 | 1.48 | <0.01 |
Y2 | CatZ | Q9WUU7 | 1.79 | 0.04 | 1.08 | 0.85 |
Y3 | Ncf4 | P97369 | 1.15 | 0.02 | 1.19 | 0.01 |
Enzyme | Control | Acute | Recov | Ctrl + silver ion |
---|---|---|---|---|
All the activities are expressed in nmol substrate converted per min per mg total protein. Statistical significance of the results in the Student T test: *p < 0.05. ND: not determined. Abbreviations: LDH: lactate dehydrogenase; MDH: malate dehydrogenase; TPIS: triose phosphate isomerase; BVR: biliverdine reductase; PDXK: pyridoxal kinase; GAPDH: glyceraldehyde phosphate dehydrogenase; 6PGDH: 6-phosphogluconate dehydrogenase; PNPH: purine nucleoside phosphorylase; HXK: hexokinase; ENO: enolase; IDHC: NADPH-dependent isocitrate dehydrogenase. | ||||
LDH | 74.4 ± 2.75 | 68.9 ± 1.14* | 73.8 ± 4.13 | ND |
MDH | 37.4 ± 0.95 | 31.3 ± 2.85* | 31.7 ± 2.36* | ND |
TPIS | 70.7 ± 8.75 | 76.6 ± 5.58 | 76.3 ± 7.80 | ND |
BVR | 0.67 ± 0.04 | 0.53 ± 0.1* | 0.79 ± 0.06* | ND |
PDXK | 7.4 ± 0.95 | 5.9 ± 0.6* | 7.5 ± 0.68* | ND |
GAPDH | 157.12 ± 14.5 | 144.25 ± 9.77 | 157.44 ± 5.91 | ND |
6PGDH | 30.69 ± 3.17 | 39.37 ± 4.92* | 38.75 ± 1.24* | ND |
PNPH | 8.97 ± 1.17 | 9.12 ± 1.6 | 9.06 ± 0.77 | ND |
HXK | 23.4 ± 3.04 | 16.3 ± 4* | 23.3 ± 2.45 | (1 μM) 14.2 ± 2.93 |
ENO | 214.5 ± 13.6 | 166.4 ± 28.1* | 184 ± 41 | (10 μM) 167 ± 12.4 |
IDHC | 16.37 ± 1.48 | 17.62 ± 2.79 | 23.75 ± 2.97** | (5 μM) 8.06 ± 1.09 |
We thus measured the glucose consumption during the last 36 hours of the experiments by measuring the remaining glucose in the culture medium at the end of the experiment. The initial medium contained 4.1 g of glucose per liter due to the dilution brought by the addition of fetal serum to obtain the complete medium. In the untreated, control cells, 1.8 ± 0.2 g of glucose per liter remained in the culture medium after 36 hours of culture. In contrast, 1 ± 0.05 g of glucose and 0.9 ± 0.04 g of glucose remained in the medium for the acute and recovery conditions, respectively. These differences were statistically significant (Mann Whitney U test, p < 0.05).
The increased glucose consumption in the acute exposure condition could be correlated with the less efficient mitochondria via a Warburg effect. However, this correlation did not hold for the recovery condition, in which the mitochondria appear as efficient as those of the control cells. This may suggest that the silver expulsion process that takes place during the recovery period consumes a lot of energy.
Phagocytosis is one of the specialized functions of the macrophages that is highly dependent on the actin cytoskeleton. We thus tested the phagocytic activity of the cells. The results, shown in Fig. 8, indicated that the proportion of phagocytic cells does not change upon treatment with silver nanoparticles. However, the number of internalized particles per cell, as described by the mean fluorescence, slightly decreased immediately after exposure to nanoparticles and went back to quasi-normal values after 72 hours of recovery.
Ctrl | Acute | Recov | |
---|---|---|---|
All the concentrations are expressed in nmol nucleotide per mg total protein. | |||
NADPH | 1.87 ± 0.12 | 1.88 ± 0.03 | 1.85 ± 0.01 |
NADP + NADPH | 2.5 ± 0.09 | 2.33 ± 0.04 | 2.62 ± 0.06 |
NADPH/(NADP + NADPH) | 0.75 ± 0.07 | 0.81 ± 0.02 | 0.71 ± 0.02 |
Most of the toxicological studies on nanoparticles use an acute exposure scheme; they expose cells to a high, but non-lethal dose. Such a scheme does not represent chronic daily exposure under, for example, occupational conditions, but may represent the dose following an accidental exposure. To fully evaluate the toxicity of AgNPs, it is therefore important to know not only the acute effects post exposure, but also how cells recover from such exposures to high dosages of AgNPs. To accomplish this, a ternary comparison must be made between cells before and immediately after treatment and also between cells at the end of a recovery period. In this experimental frame, the acute exposure point is not the primary focus of attention. We therefore decided not to investigate in detail the role of silver ions in the acute response, based on the fact that the rather large silver nanoparticles used in this study dissolve to a very low extent,23 producing free silver ion concentrations that are far below the LD20 observed for silver ions. Furthermore, this aspect has been investigated in numerous studies (e.g. in ref. 8, 15, 59 and 85) and the general outcome of these studies is that the mechanisms observed for silver nanoparticles cannot be explained by silver ions, at least for large nanoparticles.15
In this frame, the proteomic approach is of interest because it can be used to explore not only a few parameters, as targeted approaches do, but also can be used to investigate a few hundred parameters at the same time, i.e. the abundances of the various proteins analyzed in a proteomic screen. In this respect, the use of a 2D gel-based proteomic approach may appear as a medium scale approach, compared to deep shotgun proteomic approaches. However, compared to shotgun proteomics, 2D gels have the unique ability to separate protein forms, without the requirement that they appear as a single product. With growing recognition of the importance of post-translational modifications, the ability to do this is biologically relevant. Indeed, we have shown both in this study and in previous ones53,86 that some enzyme activities correlate with a single protein form and not with the sum of all the protein forms, showing the relevance of this parameter.
First, we performed a global analysis of the proteomic results. Both the principal component analysis and the analysis of similarity (ANOSIM) indicated that the recovery state was not just an intermediate state between the unexposed cells and the acutely-exposed cells. If this were the case, the recovery state should not have been separated from the other two states as it is in the principal component analysis. This is further confirmed by ANOSIM. The fact that all the p-values in the binary comparison are low and rather similar shows that the three states are significantly different from each other. If the recovery stage was just between the other two stages, at least one of the p-values between the recovery stage and one of the other two stages should be much higher than the p-value between the control and the acute stage. As this is not the case, we can reject the hypothesis that the recovery phase is only an intermediate between the control and acutely-treated stages.
We then performed a more detailed analysis of the proteomic results. The first important challenge was to determine to what extent the changes observed through proteomics are specific to silver nanoparticles and which are due to the simple presence of a PVP-coated nanoparticle. We have addressed this question in previously published studies.35,53 In these studies, we showed that PVP-coated zirconium oxide nanoparticles induced minimal changes at the proteomic level. This ruled out the possibility that the changes that we observe in the present study could be due either to the internalization process per se or are due to the effect of the addition of PVP.
As a further step, we compared our results obtained immediately after exposure to those published in the literature.9,10,24 It should be noted that this comparison is challenging because these other studies did not use the same cell type as was used here (although two of them use human intestinal cell lines, however different ones9,24) or the same silver nanoparticles. Citrate-coated silver nanoparticles were used in one study,24 while the other two used surfactant-coated nanoparticles,9,10 and in this study PVP-coated nanoparticles were used. These differences are very likely to explain the wide differences observed between the studies in which proteins were differentially modulated by treatment, with a minimal overlap between the results. Despite this general trend, several convergences could be observed between our study and those previously published. For example, increases in the abundances of Ran, Moesin, RuvB2, EfhD2, Pfkl, Anxa3, Tcpa and Tbcb were observed both in our study and the one published by Verano-Braga et al.24
However, proteomics is prone to multiple testing issues, so that the results obtained by proteomics must be verified by independent, targeted experiments. To accomplish this, we analyzed enzyme activities. Our enzyme activity results were consistent with observations made about changes at the proteomic level. However, a few discrepancies occurred, which were always of the same type: increases in the protein level by proteomics, corresponding to stable or decreased activities. We could, however, show that these enzyme activities are very sensitive to the silver ion, which was released within cells during their exposure to silver nanoparticles. Thus, the increase in protein levels can be seen in such cases as a cellular mechanism to compensate for the decrease of activity brought by silver.
We then carried out indirect validation studies, for example on the free glutathione levels, which were decreased immediately after exposure, a phenomenon that was previously observed for copper nanopartlcles.35 We also tested phagocytosis, which is an important function of macrophages and is important for clearing bacteria. For this assay we used micron-sized fluorescent beads, which functioned as bacterial mimics. This allowed us to investigate whether nanoparticle-treated cells were still able to clear bacteria. The proteomic screen suggested that phagocytosis might be altered after treatment with silver nanoparticles, which was confirmed in our assay testing the ability of the cell to clear bacteria. These results were previously observed for primary macrophages.23
The key question asked in this study, however, does not revolve around the acute response to silver nanoparticles, but instead asks to what extent and by which means do cells recover after such an exposure. In a simple model for recovery, protein changes should exhibit trends that return to normal, i.e. the amplitude of the change in abundance between the recovery and control stages should be lower than the amplitude of the change in abundance between the acute exposure and control stages. Out of the 239 spots that are highlighted in the proteomic screen, 135 (56%) show such a trend. This means that 104 (44%) show a stronger response at the recovery phase than immediately after exposure. With such a split trend, it is interesting to evaluate the results of targeted experiments during the recovery phase.
Interestingly, this split trend was also apparent in the results of targeted experiments. General metabolic processes seem to return to normal (mitochondrial potential, glutathione levels, and most enzymatic activities), as well as phagocytosis and basal NO production (without LPS stimulation). Oppositely, the LPS-induced activities (e.g. NO, IL-6 and TNF production) worsen during the recovery phase, as well as the isocitrate dehydrogenase activity, and the glucose consumption, which stays much higher at both the recovery and acute exposure phases than in unexposed cells.
It was the necessary to check in detail how proteomic changes correlate with observed functional changes. For example, for mitochondrial proteins, the proteomic screen detects an increase in the beta subunit of ATP synthase (ATPB) and an increase in one subunit of respiratory complex I (NDUV2) upon cell exposure to silver nanoparticles. At the same time the mitochondrial transmembrane potential decreases slightly, showing that this increase in mitochondrial proteins could be an attempt to restore normal energy production in cells. Upon recovery, one of the ATPB spots slightly decreases compared to the value immediately after exposure, while the other spot further increases. The NDUV2 spot is also enhanced. However, even the ATPB spot that decreased is still significantly more abundant at this stage of recovery than in control cells. This shows that the cellular situation is still not back to normal, as can be expected from the still high intracellular silver content, and that restoring the metabolic activity of a cell is an active process requiring an upregulation of several proteins.
Opposite to the delayed return to control values, GCLM, for example, shows an increase just after exposure. When the glutathione demand is high and the free glutathione levels are low, there is a return to control values at the end of the recovery period, at which point free glutathione levels are close to normal again.
In contrast to this simple case, the regulation of the actin cytoskeleton is much more complex. Many of the proteins interacting with actin and regulating its dynamics are regulated by phosphorylation. This holds true for cofilin,74 actinin 4,87 vinculin,88 swap70,89 arpc5,90 gelsolin91 and Arp2.92 While this further demonstrates the interest of taking into account modified protein forms, there are no obvious rules to predict which modified spot(s) is (are) effector(s) on the actin cytoskeleton. For example, the effector spot for cofilin is the median spot.93 Consistent with what has been described in the literature, we observe mostly modulations on acidic, modified forms of the above-cited proteins.
In line with the sustained changes in the actin cytoskeleton observed even at the recovery stage, we observe sustained changes in these modified spots, as exemplified by spots 2 of actinin 4 and cofilin, and spots 1 for Arp2 and Arpc5. It must be recalled, however, that such changes are not induced by the particle internalization process per se, as they were not observed for cells treated with the non-toxic zirconium oxide nanoparticles.53 They are also not induced by every toxic nanoparticle, as they were observed neither with zinc oxide53 nor with copper oxide, except for arpc5 which also decreased in response to treatment with copper oxide.37
Overall, the proteomic results suggest that the recovery process is a slow process. To evaluate this aspect, we determined the number of spots that showed a fast recovery. This category was defined as spots for which the quantitative change in the acute vs. control comparison showed an amplitude at least twice that in the recovery vs. control comparison. Only 44 spots were identified that fell in this category (including GCLM).
A striking result of our study was the observation that some changes in protein levels that were not immediately significant after exposure became significant at the end of the 72 h recovery phase. Such changes were observed both in targeted experiments (e.g. for the LPS-dependent responses) and in the proteomic screen. 51 spots showed such an expression profile, i.e. 21% of the total variable spots.
Functionally speaking, the results obtained when evaluating LPS-induced activities contrast with those previously described on primary macrophages,23 in which the cytokine and TNF productions also tended to return to normal values during the recovery phase. This discrepancy may have several origins. One such cause for the discrepancy could be differences in the persistence of silver between differing experimental conditions. In the experiments on primary macrophages, measurements of silver by PIXE showed a strong decrease (50%) in cellular silver content upon recovery, without any cellular multiplication (primary macrophages are post-mitotic in vitro). In the current experiments using the J774 cell line, the total silver content decreased by only 15%, and the decrease in cellular silver content is due mostly to cell division. The simple fact that the cells are able to divide shows that the silver concentration present in the cells is not toxic and explains also why, generally, the cellular parameter tends to return to normal levels. The remaining silver concentration may, however, may be high enough to inhibit specialized macrophage functions that depend on cell signaling, such as LPS-dependent activities.
In this context, it is tempting to dismiss the results obtained on the cell lines and favor those obtained using primary cells. The situation may, however, not be so simple. First, primary macrophages in vitro survive for only a few days once differentiated, while resident macrophages, such as those used in experiments using cell lines, have a very long lifespan,94,95 Furthermore, the condition in which the results between cell lines and primary macrophages diverge corresponds to an acute exposure to silver nanoparticles followed by a massive exposure to bacteria (mimicked by exposure to LPS), i.e. conditions that have not been tested in vivo. Therefore, we are currently unable to determine which system best represents the in vivo situation.
Footnotes |
† Electronic supplementary information (ESI) available. See DOI: 10.1039/c9en00408d |
‡ These authors contributed equally to this work. |
§ Deceased: Nov. 21st, 2018. This paper is dedicated to her memory. |
This journal is © The Royal Society of Chemistry 2019 |