S. Kavaliauskiene et alSupplementary 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.
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.
Table S1 Lipid content in HEp-2 cells grown for 1, 2 or 3 days in the culture
LIPID CLASS / 1 day / 2 days / 3 dayspmol/µg protein / mol% of total / pmol/µg protein / mol% of total / pmol/µg protein / mol% of total
PC / 27.6 / 30.9 / 33.7 / 33.2 / 35.8 / 32.5
PC O/PC P / 5.00 / 5.6 / 4.88 / 4.8 / 5.53 / 5.0
PE / 15.5 / 17.3 / 17.1 / 16.8 / 16.4 / 14.9
PE O/PE P / 5.11 / 5.7 / 5.72 / 5.6 / 5.47 / 5.0
PS / 14.8 / 16.5 / 17.2 / 16.9 / 19.4 / 17.6
PI / 1.78 / 2.0 / 1.71 / 1.7 / 1.74 / 1.6
PA / 0.45 / 0.5 / 0.93 / 0.9 / 1.58 / 1.4
DAG / 1.23 / 1.4 / 1.42 / 1.4 / 1.74 / 1.6
CE / 5.44 / 6.1 / 3.86 / 3.8 / 5.86 / 5.3
SM / 7.51 / 8.4 / 9.05 / 8.9 / 9.66 / 8.8
Cer / 0.60 / 0.7 / 0.71 / 0.7 / 0.87 / 0.8
GlcCer / 0.23 / 0.3 / 0.29 / 0.3 / 0.32 / 0.3
LacCer / 0.84 / 0.9 / 1.10 / 1.1 / 1.00 / 0.9
Gb3 / 1.48 / 1.7 / 2.03 / 2.0 / 2.46 / 2.2
LPC / 0.87 / 1.0 / 1.09 / 1.1 / 1.25 / 1.1
PG / 0.68 / 0.8 / 0.63 / 0.6 / 0.83 / 0.8
LPE / 0.31 / 0.3 / 0.27 / 0.3 / 0.21 / 0.2
TOTAL / 89.4 / 100 / 101.7 / 100 / 110.2 / 100
Abbreviations of all lipid classes are given under the section “Annotations of lipid species” in Methods.
Fig. S4Chain 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 poly-unsaturated (PUFA) FAsin 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 mono-unsaturated, 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 inthe group 0:1). The same colouringis used for graphs a, b and c. (d)Distribution of species withdifferent saturation patterns in each glycerolipid class. Abbreviations of all lipid classes are given under the section “Annotations of lipid species” in Methods.
Fig. S5Stx-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 endocytosedStx 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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