Electronic Supplementary Material (ESI) for Nanoscale.
This journal is © The Royal Society of Chemistry 2016
Supplementary Information
to
Metal homeostasis disruption and mitochondrial dysfunction in
hepatocytes exposed to sub-toxic doses of zinc oxide nanoparticles
M. Chevalleta, B. Galletb, A. Fuchsc, P.H. Jouneaud, K.Uma, E. Mintza, I. Michaud-Soreta
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Figure S1. Size distributions of a) ZnO-NP-FBS and b) ZnO-NP-silane preparations
a)
60
50
% intensity
40
30
20
10
0
0,1
1
10
100
1000
diameter (nm)
b)
50
% intensity
40
30
20
10
0
0,1
1
10
100
1000
diameter (nm)
2
Figure S2. Measurement of SOD and CAT activities after a 6 hour incubation with Zn acetate, ZnO-NP-FBS or
ZnO-NP-silane at 90 µM. Results were expressed in enzymatic units/mg of protein as the average + SEM of 4
independent experiments. P>0.05
16
control
ZnO-NP-silane
ZnO-NP-FBS
Zn acetate
SOD activity (U/mg protein)
14
12
10
8
6
4
2
0
CAT activity (U/min/mg protein)
25
20
15
10
5
0
3
Figure S3. Quantification of acidic vesicular organelles with acridine orange staining. Results are presented as
changes in mean of green fluorescence expressed as a percentage of the mean fluorescence in control cells
(three independent experiments). Fluorescence measurements were carried out on a MoFlo cell sorter. ***:
p<0.001
control
ZnO-NP-silane
ZnO-NP-FBS
Zn acetate
Mean fluorescence intensity (%)
140
***
120
***
***
100
80
60
Figure S4. Quantitative PCR analysis of DRP1, MFN1 and MFN2 mRNA expression in HepG2 cells after a 6 hour
incubation with Zn acetate, ZnO-NP-FBS or ZnO-NP-silane at 90 µM. Results are presented as relative
expression changes after normalizing with HPRT and 36B4 mRNA. Each value represents the mean of relative
expression +SEM of three independent experiments. P>0.05
control
ZnO-NP-silane
ZnO-NP-FBS
Zn acetate
1,8
1,6
Fold increase
1,4
1,2
1,0
0,8
0,6
0,4
0,2
0,0
DRP1
MFN1
MFN2
4
Table S1. Quantitative real time polymerase chain reaction primer sequences
gene
Met1X
HMOX1
HSPA6
SOD1
SOD2
CAT
ZnT1
ZnT2
ZnT7
Zip1
Hamp
GCLM
EGR1
MTF1
DRP1
MFN1
MFN2
HPRT
36B4
Forward
Reverse
GCT TCT CCT TGC CTC GAA A
TGA CGT CCC TTT GCA GAT G
ATG ACA CCA AGG ACC AGA GC
AGG AGG TGG AGA GGA TGG TT
TGG CCG ATG TGT CTA TTG AA
GTG GAG AAC CCA AAG GGG AG
TTG CCA CAG GAA AGT ACC CC
CGC AGC CAT GGG GTG TTG GG
AAC TTT GGC TGG CAG AGA GC
GTG CCT GAA CCT CTC TTT CG
GCC AGG AGC TAA CCA TGA AG
ACC AGT GGC TCT GTT TTC CC
CCT CCT GCT GTG TGA TGC CAC C
TAC GAG CAC CTG ACC GCA GAG TC
CGA AGG AGA AGC CAT TTG AG
TCA CCC GGA GAC CTC TCA TT
TGT TTTGGT CGG AAA CTC TG
TAT CCA CTT CCC TCC TCC CG
ATG GAC AGG ACT GAA CGT CTT GCT
GAA ATC CTG GGT GTC CGC AAT GTT
GTG TAA GGA CCCA TCG GAG A
TGT CCT CTT CGG GAA TCT TG
ACC TTT GCC CAA GTC ATC TG
GAA ACC AAG CCA ACC CCA AC
TGA GGC CAA ACC TTG GTG AG
CAT CGC CAG CGA CGA GGT CA
TGG TGA AGG GTC AAC CCC AT
CGG AAA TCA GCC TAA GCA G
ATG GCC AGG ATG AAC TCT TG
CAG ATG GGG AAG TGG GTG TC
CGT GCG CTT GAA TGT CAG GAA TGC
CAT GAA GCT CCT CGC TGT GCG TG
ATT TGC TGC AGC CTT CAG AT
TCT GCT TCC ACC CCA TTT TCT
CTGTCTGCGTACGTCTTC CA
CCACCAGTCCTGACT TCA CC
TTG AGC ACA CAG AGG GCT ACA ATG
AGA CAA GGC CAG GAC TCG TTT GTA
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