S. Kavaliauskiene et al
Supplementary material 1
Fig. S1 Distribution of cell cycle phases is not different in low and high density HeLa cells
(a) Low and high density HeLa cells were incubated with 2 µg/ml Hoechst 33342 in complete culture
medium for 30 min at 37ºC and analysed by flow cytometry. Inserts: phase contrast images of the cells.
(b) Distribution of cell cycle phases in low and high density cells. The figure shows average percentage
of cells in each cell cycle phase. The error bars represent the difference between the average value and
the values obtained from two independent experiments. Quantification was performed using FlowJo
7.6 software.
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Supplementary material 1
Fig. S2 The rate of protein synthesis per cell is lower in high density cells
Low and high density HeLa cells were incubated for 3 hours in leucine-free medium prior to addition
of 1 µCi/ml [3H]leucine. The incubation was then continued for 20 min, and the amount of
incorporated [3H]leucine was measured by β-counting. The rate of protein synthesis was quantified as
cpm/cell, and presented as percentage of low density samples. The graph shows mean values + SEM, n
= 3, *p < 0.05, paired Student t-test.
Fig. S3 High density HEp-2 cells are less sensitive to Shiga toxin (Stx)
Low and high density HEp-2 cells were treated with ten-fold serial dilutions of Stx for 3 hours in
leucine-free medium. Cells were then incubated in the presence of [3H]leucine for 20 min, and protein
synthesis was measured. (a) One representative experiment is shown; error bars represent difference
between the mean value and two parallels. (b) The quantification of IC50 for low and high density cells.
The graph shows mean values + SEM, n = 3, *p < 0.05, paired Student t-test.
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Supplementary material 1
Table S1 Lipid content in HEp-2 cells grown for 1, 2 or 3 days in the culture
LIPID CLASS
PC
PC O/PC P
PE
PE O/PE P
PS
PI
PA
DAG
CE
SM
Cer
GlcCer
LacCer
Gb3
LPC
PG
LPE
TOTAL
1 day
pmol/µg mol%
protein
of total
27.6
30.9
5.00
5.6
15.5
17.3
5.11
5.7
14.8
16.5
1.78
2.0
0.45
0.5
1.23
1.4
5.44
6.1
7.51
8.4
0.60
0.7
0.23
0.3
0.84
0.9
1.48
1.7
0.87
1.0
0.68
0.8
0.31
0.3
89.4
100
2 days
pmol/µg mol%
protein
of total
33.7
33.2
4.88
4.8
17.1
16.8
5.72
5.6
17.2
16.9
1.71
1.7
0.93
0.9
1.42
1.4
3.86
3.8
9.05
8.9
0.71
0.7
0.29
0.3
1.10
1.1
2.03
2.0
1.09
1.1
0.63
0.6
0.27
0.3
101.7
100
3 days
pmol/µg mol%
protein
of total
35.8
32.5
5.53
5.0
16.4
14.9
5.47
5.0
19.4
17.6
1.74
1.6
1.58
1.4
1.74
1.6
5.86
5.3
9.66
8.8
0.87
0.8
0.32
0.3
1.00
0.9
2.46
2.2
1.25
1.1
0.83
0.8
0.21
0.2
110.2
100
Abbreviations of all lipid classes are given under the section “Annotations of lipid species” in Methods.
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Supplementary material 1
Fig. S4 Chain length and saturation of the fatty acyl chains of lipids in HEp-2 cells
(a) The chain lengths of fatty acyl chains (FA) of lipids from HEp-2 cells grown for 1, 2 or 3 days. (b)
Total content of saturated (SFA), mono-unsaturated (MUFA), di-unsaturated (DUFA) and polyunsaturated (PUFA) FAs in the lipids. (c) Saturation of glycerolipid species: 0:0 – both FAs are
saturated, 0:1 – one FA is saturated, and the other is mono-unsaturated, 1:1 – both FAs are monounsaturated, 0:2 – one FA is saturated, and the other is di-unsaturated, 0:poly – one FA is saturated, and
the other is poly-unsaturated, 1:2 – one FA is mono-unsaturated, ant the other is di-unsaturated, 1:poly
– one FA is mono-unsaturated, and the other is poly-unsaturated. The order of the FAs is not taken into
account (e.g. both saturation patterns, 0:1 and 1:0, are included in the group 0:1). The same colouring is
used for graphs a, b and c. (d) Distribution of species with different saturation patterns in each
glycerolipid class. Abbreviations of all lipid classes are given under the section “Annotations of lipid
species” in Methods.
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Supplementary material 1
Fig. S5 Stx-biotin endocytosis in HeLa cells is not affected by cell density
Low and high density HeLa cells were treated with 42 ng/ml biotin labelled Stx (Stx-biotin) for 20 min
at 37ºC. Half of the samples were then treated with 0.1 M MESNA in HEPES buffer (pH 8.6) to reduce
the disulfide bond between Stx and biotin, and thus remove the biotin from cell surface-bound but not
internalised Stx-biotin. Finally, both MESNA-treated and non-treated cells were washed and lysed. For
the detection of Stx-biotin, Stx-biotin was labelled by streptavidin coated magnetic beads
(Dynabeads®, Life Technologies) and BV-TAG® linked antibodies (BioVeris Corporation), and
detected using M-SERIES® M1R analyser (BioVeris Corporation). The endocytosed Stx was
quantified as percentage of total cell associated toxin. The graph shows mean values of two
independent experiments. The error bars represent the difference between the mean value and the
values obtained from each individual experiment.
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