Download Supplementary Figure Legend (doc 33K)

Legends for Supplementary Figures
Supplementary Figure S1. TCTP is expressed within MVB of apoptotic EC. (a) Electron
micrographs.
EC
fixed
in
PLP
and
embedded
in
Lowicryl.
Protein
A-gold
immunolabelling for TCTP. The gold particles (5 nm, arrows) are present within the large
MVB. Numerous small vesicles are delineated within these bodies (arrowheads). Bar:
0.25 μm. (b) Electron micrographs. Upper panel: EC cultured in normal medium or in
serum-free medium for 4 hours, fixed in glutaraldehyde, postfixed in osmium tetroxyde
and embedded in Epon. The left upper panel (Normal EC) illustrates a small
multivesicular body. Bar: 0.25 μm. The right upper panel (Apoptotic EC) demonstrates
numerous vesicles in a large MVB. Bar: 0.5 μm.
Lower panel: morphometrical
evaluation of MVB. Electron micrograph fields of the cell cytoplasm were recorded at
x7000 and enlarged at x21000. The volume density of the multivesicular bodies was
evaluated in relation to the cell cytoplasm (nuclei were not included in the evaluation) by
direct planimetry using the Zeiss Videopan software (MVB area/cell area um2).
50
cytoplasmic fields were evaluated for each condition, *= p = 0.001. The volume density
of MVB increases by more than 7 fold during serum starvation. (c) Exosome-like
nanovesicles secreted by apoptotic EC inhibit apoptosis of VSMC. Percentage of
apoptotic cells as defined by increased chromatin condensation without cell membrane
permeabilization (as evaluated with HO and PI staining) in VSMC grown to confluence in
24 well plates and exposed for 24 hours to 0.5 ml of: normal medium (N); serum
starvation in RPMI (SS); un-fractionated serum-free medium conditioned by apoptotic
EC (Total); exosomal fraction (Exo) purified by ultracentrifugation (100 000 x g 18 h) of
serum-free medium conditioned by apoptotic EC (12 ml) and resuspended in 12 ml of
RPMI (SS); the residual exosome-free supernatant (SN); exosomal fraction and
exosome-free supernatant recombined together after ultracentrifugation (SN + Exo). * =
p ≤ 1 x 10-5 vs. SS, & = p ≤ 1 x 10-5 vs. Total, n ≥ 12. (d) Amino acid sequence of human
TCTP. The diagram shows the complete amino acid sequence of human TCTP (1-172;
http://www.uniprot.org/uniprot/P13693, peptides identified by LC-MS/MS denoted by * in
Figure 2d (dash lined), amino acid sequence of the partial recombinant TCTP (yellow).
(e) Quantification by immunoblotting and densitometry of TCTP released in the SSC-Apo
using recombinant TCTP for the standard curve.
1 ml of SSC-Apo (performed in
triplicate) was TCA-Precipitated followed by immunoblotting for TCTP.
Representative
of 3 independent experiments. (f) Caspase activation favors TCTP export in different cell
types. Upper panel: Percentage of apoptotic cells as defined by increased chromatin
condensation without cell membrane permeabilization (as evaluated with HO and PI
staining) in VSMC exposed to 0.5 ml of: normal medium (N); serum starvation in RPMI
(SS) for 24 h or incubated with either ZVAD-FMK (SS-ZVAD) or vehicle (SS-DMSO)
prior to SS for 24 h. * = p = 0.02 vs. SS-DMSO, n = 6. Lower panel: Immunoblots for
TCTP in exosome-like nanovesicle extracts purified from 25 ml of serum-free media
conditioned by equal numbers of VSMC treated with vehicle (Exo-DMSO) or ZVAD-FMK
(Exo-ZVAD), as described in the upper panel. 25 ug of EC cytosolic extracts were used
as positive control for TCTP. Representative of 2 experiments.
Supplementary Figure S2 Caspase inhibition and TCTP release
(a) Percentage of apoptotic cells as defined by increased chromatin condensation
without cell membrane permeabilization (as evaluated with HO and PI staining). EC
were incubated in SS with different concentrations of ZVAD-FMK or DMSO (vehicle) for
4 hours. = p ≤ 0.05 vs DMSO, n = 3. (b) Immunoblots for TCTP (left panel) in 1 ml of
total serum-free media conditioned by equal numbers of serum starved EC in presence
of ZVAD 30 uM or DMSO (Vehicle).
Right panel: quantification of TCTP from
Immunoblots analyzed by densitometry; n= 3. p ≤ 0.05 vs DMSO.
Supplementary Figure S3. Densitometry analysis for western blots of proteins
extracted from conditioned media presented in Figures 3b, 4a, 5d, 6c and g,
Supplementary Figure S1f.