SUPPLEMENTARY INFORMATIONDOI: 10.1038/NNANO.2011.188

NATURE NANOTECHNOLOGY | www.nature.com/naturenanotechnology 1

Signalling of DNA damage and cytokines across nanoparticle exposed cell barriers depends on barrier thickness A. Sood, S. Salih, D. Roh, L. Lacharme-Lora, M. Parry, B. Hardiman, R. Keehan, R.

Grummer, E. Winterhager, P.J. Gokhale, P.W. Andrews, C. Abbott, K. Forbes, M.

Westwood, J. Aplin, E. Ingham, I. Papageorgiou, M. Berry, J. Liu , A.D. Dick, R.J. Garland,

N. Williams, R. Singh, A.K. Simon, M. Lewis, J. Ham, L. Roger, D.M. Baird, L.A.

Crompton, M.A. Caldwell, H. Swalwell, M. Birch-Machin, G. Lopez-Castejon, A. Randall,

H. Lin, M-S. Suleiman, H. Evans, R. Newson, C.P. Case

SUPPLEMENTARY INFORMATION

METHODS

Corneal cells

HCE-T corneal epithelial cells were used to provide monolayered and bilayered corneal

barriers as they are most commonly used human corneal epithelial cells in this respect1,2.

These cells have been immortalised with a recombinant SV40-adenovirus vector.

Telomerase activity was detected using the TRAPeze real time PCR kit (Chemicon

international, Billerica, MA, USA). 1x106 CLL B-cells were lysed in 200 µl CHAPS buffer

and each lysate was included in the TRAPeze reaction using the Amplifluor primers and

Titanium Taq (BD Clontech, Mountain View, CA, USA). Each reaction was carried out in

triplicate in 96 well plate format, including positive control extracts, positive PCR controls

that generated the standard curves for each plate, heat treated controls and a no template

control. Telomerase activity was expressed as Log copy number calculated using the Ct

values and the standard curve for each 96 well plate. Telomere lengths were measured using

STELA3.

Analysis of 8-oxodG in cellular DNA

DNA was extracted from frozen cell pellets using a modified chaotropic procedure, the Wako

Sodium Iodide (NaI) extractor kit (Wako Chemicals GmbH, Neuss, Germany) as described

previously4. The levels of 8-oxodG adducts were determined in the cellular DNA samples

using positive electrospray ionization online column-switching liquid chromatography-

tandem mass spectrometry (LC-MS/MS) selected reaction monitoring (SRM) with the

incorporation of a stable-isotope internal standard for 8-oxodG as described previously4.

Ca imaging

Standard microfluorometric imaging of intracellular Ca2+

concentration ([Ca2+

]i ) was

performed using the Ca2+

-sensitive fluorescent indicator Fura2, as described previously5. For

all imaging experiments either fibroblasts or BeWo trophoblasts were cultured on 13 mm

diameter round glass coverslips. BJ Fibroblasts were plated at a density that allowed cell by

cell analysis in our imaging experiments. In contrast, for most experiments, BeWo cells were

grown to confluent layers 1 or 2 cells thick- although in a few additional comparative

experiments measurements were made from BeWo cells grown at lower densities. A single

glass coverslip bearing Fura2 loaded cells was transferred to a custom low-volume chamber

(Warner Instruments), which was in turn mounted on the stage of an inverted fluorescence

microscope (Nikon TE2000). HBSS bath solution was applied to the preparation at ~2

mLmin-1

by means of a peristaltic pump and was subsequently aspirated to waste using a

vacuum pump. All drugs were bath applied in the circulating HBSS. During a few

experiments a Ca2+

-free version of this solution was applied to cells to examine [Ca2+

]i

responses arising solely from Ca2+

release from intracellular stores. Standard ratiometric

imaging of [Ca2+

]i was performed at room temperature using a fast switching Sutter DG4

light source and a Hamamatsu Orca 12 AG camera. The individual images from each time

point were background corrected and thresholded before the 340:380 ratio was calculated-

this ratio is proportional to [Ca2+

]i.

Connexin immunostaining

BeWo cell barriers were fixed in 3% paraformaldehyde (PFA) in PBS for 20min at RT.

Blocking was performed for 1hr at RT with 10% heat-inactivated goat serum in PBS. Cells

were incubated overnight with rabbit polyclonal antibodies Gap 11, Gap 5+6, Gap 7M

(diluted 1:200) targeting connexin extracellular loops as previously characterized6,7

.

Following three 10min PBS washes, cells were incubated for 1hr at RT with goat anti-rabbit

Alexa Fluor 488 antibody (1:2500, Invitrogen) and DAPI (0.5ug/ml, Sigma). Images were

acquired on an Olympus FluoView 300 laser-scanning confocal microscope using 40x and

60x oil objectives.

Western blotting

Western blotting analysis was used to investigate activation of the signal transduction

molecules, ERK, Akt, mTOR and HIF1α. Barrier BeWo cells, both monolayers and bilayers,

before and after exposure to CoCr nanoparticles for up to 24 hr were lysed in Laemlli buffer

containing sodium orthovanadate, PMSF, chymostatin, leupeptin, antipain and pepstatin A.

Lysates were electrophoresed in either 7, 8 or 10% (w/v) polyacrylamide and proteins

transferred onto PVDF membranes and incubated overnight at 4°C with the appropriate

antisera. Secondary anti-rabbit IgG conjugated to horseradish peroxidase were applied for 1hr

at room temperature and proteins were visualised with ECL Plus reagent.

Human embryonic stem cells

hESCs were manually passaged using glass needles from feeder culture plates onto 13mm

coverslips pre-coated with Matrigel™ (BD Biosciences) in wells containing 1.5 ml of growth

media. Cells maintained as undifferentiated were cultured in mTeSR™1 media (Stem Cell

Technologies), also containing streptomycin (10mg/ml), penicillin (10,000U/ml) and

amphotericin B (250µg/ml) (all Invitrogen). Cells that were allowed to differentiate were

cultured in regular growth media (as previously described) without FGF-2 and supplemented

with addition of 10M SU5402 (Calbiochem), an FGF-2 inhibitor to initiate their

differentiation. Approximately 20 colony pieces were plated per coverslip.

After 24 hours a complete media change was carried out on the hESCs and from the

differentiating cells the FGF inhibitor SU5402 was also removed. The prepared Transwell®

inserts with the BeWo cells and the cobalt chromium nanoparticles were then added to the

wells containing hESCs. In each experiment 3 wells were prepared for each condition.

After a subsequent 24 hours the barriers were removed and the hESCs were fixed with 4%

paraformaldehyde.

Further differentiation of stem cells with and without exposure was determined by staining

using anti-A2B5, (1:5, Centre for Stem Cell Biology), or anti-CXCR4, (1:100, ab2074,

Abcam) Antibodies. These were detected using FITC and Dylight-649 conjugated secondary

antibodies respectively. Nuclei were counterstained using Hoechst 33342 (Invitrogen).

qPCR of mitochondrial DNA (mtDNA) damage

mtDNA damage was measured as amplification efficiency for a large (11,095 bp) amplicon

normalized to a short (83 bp) amplicon, performed using real-time PCR as previously

described8,9

. Briefly, the PCR was performed in 50 µl reactions containing: 200ng template

DNA, 10mM buffer, 10µM of each primer, 5x Sybr green, 10mM dNTP, 3.75 units of

Expand Long Template enzyme (Roche) and made up to the required volume with high grade

PCR water. The primers used were D1B (5’-ATG ATG TCT GTG TGG AAA GTG GCT

GTG C-3’) (282-255 nt) and OLA (5’-GGG AGA AGC CCC GGC AGG TTT GAA GC-3’)

(5756-5781 nt). The cycling conditions were 94°C for 2 mins, 10 cycles of 15 seconds

denaturation at 94°C, 30 seconds annealing at 60°C and 9 mins extension at 72°C; then 25

cycles of 15 seconds denaturation at 94°C, 30 seconds annealing at 60°C and 8.5 mins

extension (+10 seconds/cycle) at 68°C. The qPCR was performed on a MJ Chromo4 (Bio-

Rad) PCR machine.

Mitochondrial immunoflourescence

Mitochondrial stains were performed by incubating at 37C for 30 min with either

100nM Mitotracker Green, 100nM NaO, or 5M MitoSOX Red (all from Molecular Probes,

Invitrogen) and directly analysed without fixing10

. Samples were analysed by flow cytometry

using a FACScalibur (Becton Dickinson)10

Hydrogen peroxide measurements

The level of hydrogen peroxide in the media was measured with the help of a Hydrogen

Peroxide Assay Kit (BioVision ) using the probe OxiRed and following the manufacturer's

instructions. In brief; samples were centrifuged for 15 minutes at 1000g within 30 minutes of

collection and H2O2 in the reaction mix was measured flourometrically (Ex/Em = 535/587

nm) using a micro-plate reader.

Extraction and measurements of purine and lactate:

Purine and lactate were extracted from the media below nanoparticle exposed barriers and

their concentration determined using HPLC (purine) or a commercially available kit (Sigma)

for lactate (Sigma) as described previously11,12

.

Supplementary figure 1. Validation of BeWo barriers Confocal microscopy of BeWo

barriers grown for 4 days (left hand side) and 7 days (right hand side). DAPI stained

preparations with side (a,b) and tangential (c,d) views. In a and b images 3D reconstructions

were made of z-stack images and rotated through 360o to allow labelling of cells according to

whether they touched the transwell insert (red asterisks), or were in 2nd

(blue asterisks) or 3rd

layer (green asterisks). ZO-1 and DAPI stained preparations with side (e,f) and tangential

(g,h) views.

Supplementary figure 2. Fluorescence values for dye transfer of Na flourescein across

BeWo barriers after exposure to CoCr nanoparticles or chemicals. Units as fluorescence.

Values are shown for (a) 4, 5, 6, 7 day barriers with (black bars) or without (open bars)

exposure to nanoparticles. In (b) (d) monolayers, (c) (e) bilayers. b,c) values are shown after

exposure of barriers to Rotanone (ROT) or Antimycin A (AMA) (dark grey bars) or in

control unexposed barriers (open bars) d) values are shown after exposure to CoCr

nanoparticle or ATP (black bars) with or without Gap 27 (GAP) or Compound 17 (C17)

(light grey bars) e) values are shown for CoCr nanoparticle exposure (black bars) with our

without MitoQ (MQ) or its control compound TPP (light grey bars). MitoQ or TTP applied

without nanoparticles (open bars).

Supplementary figure 3. DNA damage in fibroblasts beneath CoCr exposed BeWo

barriers compared to unexposed fibroblasts (Con). The BeWo barriers were grown at

different thickness for 7 days by altering the plating density cell number as shown. a) DNA

damage, measured with the alkaline comet assay, is expressed as mean tail moment with 95%

confidence intervals. DAPI stained preparations with b) side and c) tangential views.

Supplementary figure 4. mRNA of signalling channels. mRNA expression of connexin 43

(Cx43) P2Y1 or P2Y2 receptor and β actin (shown for reference) in BeWo barriers grown

between 4 and 7 days with (M) or without (C) exposure to CoCr nanoparticles.

Supplementary figure 5. Nanoparticles elevate [Ca2+

]i in BeWo trophoblast barriers and

trigger release of factors which raise [Ca2+

]i in fibroblasts. A) A typical experiment

illustrating the slowly developing rise in [Ca2+

]i observed when nanoparticles are added to a

trophoblast barrier. Nanoparticle treatment did not eliminate subsequent G-protein coupled

receptor-mediated [Ca2+

]i rises. B) Pooled data from 4 experiments similar to and including

that in (A). The data are plotted relative to the mean pre-stimulus ratio of each experiment. C)

A typical experiment comparing the mean [Ca2+

]i rises elicited in a fibroblast population of

27 cells by conditioned medium taken from beneath either an untreated control trophoblast

barrier or a barrier treated with nanoparticles for 24 hrs. The histogram (right) plots for a

number similar experiments showing the fraction of fibroblasts that responded to conditioned

from untreated trophoblast monolayers or trophoblast monolayers or bilayers exposed to

nanoparticles for 24 hrs. D) A typical experiment illustrating that Gap 27 treatment of

monolayers during exposure to nanoparticles results in a conditioned medium much less

effective at increasing [Ca2+

]i. The trace represents data pooled from 69 individual cells in one

experiment. The histogram (right) plots data from a number of experiments describing the

proportion of fibroblasts that responded to conditioned medium from nanoparticle treated

BeWo monolayers under control conditions or in the presence of Gap 27 or MitoQ. E) An

experiment comparing the [Ca2+

]i rises evoked by two halves of the same batch of

conditioned medium taken from below nanoparticle treated monolayers. One fraction was

treated with apyrase for 1 hr, whereas the remaining fraction was left untreated. Data are the

average of 44 individual cells analysed from one experiment.

Supplementary figure 6. ATP induces increases [Ca2+

]i in both fibroblasts and BeWo

trophoblast barriers. A) ATP induced [Ca2+

]i increases in fibroblasts previously cultured

beneath a BeWo cell monolayer for 24 hrs. Each trace represents [Ca2+

]i (plotted as 340:380

emission ratio) versus time from an individual fibroblast- note that ATP produces a [Ca2+

]i

rise only in a proportion of cells, whereas oxytocin is effective in all cells. B) Data from a

similar experiment to that in (A) but from fibroblasts grown below a BeWo bilayer for 1 day.

C) A whole field average from an intact trophoblast barrier plotting [Ca2+

]i versus time. The

response to ATP is both larger and less prone to desensitization than that elicited by ADP.

Supplementary figure 7. ROS. Measurements of ROS (fluorescence of H2DCFDA) in

BeWo cells with and without nanoparticle exposure at different times in minutes ranging

from 5 to 180 minutes or at 24 hours. Each point shows a mean and standard deviation.

Supplementary figure 8. Effects of altered oxygen. Light micrographs of BeWo barriers

grown for 4 (a) and 7 days(b) under hypoxic conditions (1%) followed by 1 day of

atmospheric oxygen.

Supplementary figure 9. Mitochondria after nanoparticle exposure. Untreated 4 day

BeWo barriers (monolayers) showed increased mitochondrial superoxides and increase of

mitochondrial mass compared to untreated 7 day barriers (bilayers) and after metal exposure

an increase in mitochondrial damage (decrease of intact non-oxidated cardiolipins) and a loss

of mitochondrial mass which was unaffected by blocking Gap junctions.

Untreated 4 day BeWo barriers were stained for (a) mitochondrial superoxides (Mitosox) or

(b) mitochondrial mass (MTG) and analysed by flow cytometry. Overlays are of replicates.

Day 4 (left panels) and day 7 (right panels) barrier cells were stained for (a) Non-nonyl-

acridine-Orange (NaO) or (b) MTG or (c) MitoSox and analysed by flow cytometry. Shown

is onerepresentative replicate out of three for BeWo barriers with and without 24 hour

exposure to CoCr nanoparticles with and without Gap 27 treatment (Gap).

Supplementary figure 10. Purine metabolites. Levels of the purine metabolites, GDP, IMP

Hypoxanthine, Xanthine, AMP and Adenosine in the culture medium below BeWo barriers

with (CoCr) and without (con) nanoparticle exposure and after 4, 5, 6 and 7 days of growth of

the barrier. Levels of H2O2 in the culture medium below 4 and 7 day BeWo barriers at

different times after nanoparticle exposure.

Supplementary figure 11. Human placenta. Light micrographs of placental villi during

first trimester (a) with a bilayered trophoblast (arrows) and at term (b) showing the

monolayered trophoblast (arrows) which separates the maternal blood from fetal blood

vessels. It is interesting with regard to the difference in signalling of monolayered and

bilayered barriers that there is an intimate proximity of fetal vessels to the monolayered

trophoblast at term (b) compared to the distance between the two during the first trimester (a).

Supplementary figure 12. Stem cell markers. Percentage of cells in colonies, which stained

positively for A2B5 (neuronal surface ganglioside, oligodendrocyte precursor) (a,b) and

TUJ1 (neural stem cell marker) (c,d) after plating out human embryonic stem cells (Shef 6)

and placing them below BeWo barriers after 3 (a,b) or 7(c,d) days of growth of the stem cell

cultures. In each figure the first three bars show values after placing them below 4 day

(monolayered) BeWo cell barriers whilst the last 5 bars show values after placing them below

7 day (bilayered) BeWo cell barriers for 24 hours Values are shown for experiments in which

the barriers were exposed to nanoparticles (NP) altered oxygen from 1% to 21% (1%) with or

without Gap 27 (Gap) or without exposure (Cont) ** p<0.01,*** p<0.001.

Supplementary figure 13. Corneal barriers. Confocal microscopy of non stratified (left

hand side) and stratified (right hand side) corneal barriers. DAPI stained preparations with

side (a,b) and tangential (c,d) views. In a and b images 3D reconstructions were made of z-

stack images and rotated to allow labelling of cells according to whether they touched the

transwell insert (red asterisks), or were in 2nd

(blue asterisks) or 3rd

layer (green asterisks).

ZO-1 and DAPI stained preparations with tangential (e, f) and side view (g) views. h) e

cadherin stained side view. The surface of the barriers is not flat (c, d) and as a result the

confocal images of tangential views of ZO-1 staining are not complete in this view (e, f).

Supplementary figure 14. Sectretion of purine metabolites and lactate from corneal

barriers. Changes in lactate secretion from monolayer (Mono) and bilayered (Bi) corneal

barriers with (NP) and without (Cont) nanoparticles exposure with and without Gap 27 (Gap),

MitoQ (MQ) and vitamin C (VitC). Lactate (µmole/L) was measured using a commercially

available kit

Changes in purine metabolite secretion from monolayered (MONO) and bilayered (unless

stated) barriers at 1 and 24 hours after nanoparticle exposure with and without Gap 27, MitoQ

and vitamin C. Purine and nucleosides (µmole/L) were measured using HPLC.

Table 1. Cytokine chemokine and growth factor secretion from barriers. Table shows

level of cytokine expression in culture media placed below BeWo barriers ( 4 day (D)

(monolayered, 6 or 7 day (D) barriers bilayered (Bi 7 day) and corneal bilayered (Bi) or

monolayered (mono) barriers after 1 hour or 24 hour of exposure to CoCr nanoparticles (NP)

(placed in the upper chamber) with or without the additional presence of Gap 27 (Gap)

MitoQ (MQ) vitamin C (Vit C).

Supplementary Figure S1

Supplementary Figure S2

Supplementary Figure S3

Supplementary Figure S4

Supplementary Figure S5

a b

c d

e

Supplementary Figure S6

Supplementary Figure S7

Supplementary Figure S8

Supplementary Figure S9

Supplementary Figure S10

Supplementary Figure S11

Supplementary Figure S12

Supplementary Figure S13

Supplementary Figure S14

Supplementary Table 1

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