Supplemental figure 1. a. Comparison of Myr-Palm-snap-LQTDG-YFP, Myr-Palmsnap3-LQTDG-YFP and Myr-Palm-snap5-LQTDG-YFP with Myr-Palm-LQTDGmCherry. Cleavages were plotted after normalization as described in supplemental
material. Intensities were normalized so that complete cleavage corresponds to 1.
While the SNAP tag allows a more efficient cleavage, its length has no apparent
effect. Scale bars: 10m. b. Western blots showing the cleavage of the different
probes (expected sizes: 31kD for NES-ELQTDG-YFP and MyrPalm-ELQTDG-YFP,
82kD for mGFP-ELQTDG-cytoketain8, 98kD for Calnexin-ELQTDG-YFP, 41kD for
MitoNEET-ELQTDG-YFP, 43kD for H2B-ELQTDG-YFP, and 28kD for the cleavage
products). Lysates were performed at different time points after induction. Probes
were recognized using a GFP antibody.
Supplemental figure 2.
a. NES-LQTDG-EYFP and NES-LQTDG-mCherry were
coexpressed in HeLa-CD95 and monitored after induction. Cleavages were plotted
after normalization with cytosolic signals at time 0. The overlap between the curves
shows that the choice of fluorescent protein does not influence the measurement. b.
Cytoplasmic intensity of cells coexpressing Calnexin-LQTDG-EYFP and NESLQTDG-mCherry. Intensities were estimated by manually segmenting the cytoplasm
of cells, measuring the intensity histogram and fitting it to a Gaussian distribution.
250 frames were acquired, corresponding to 6 hours of acquisition. Each of the
curves represents the cytoplasmic intensity for one cell. The end of the traces
corresponds to cell division. Note that no significant decrease due to photobleaching
can be observed. c. Cytoplasmic intensity of cells expressing Calnexin-MTISD-EYFP
after induction, measured like in b. The probe was not cleaved and was therefore
used as a reference for fluorescence redistribution after induction. The end of the
traces corresponds to the rounding of the cell. Note that before this rounding, the
EYFP intensity remains constant. d. Control probes. Each of the six probes without
cleavable linker remains fully intact until formation of apoptotic body with the
exception of NES-YFP that shows some nuclear signal on the last frame, likely
corresponding to export machinery disruption. e. Cleavages of the different
localization probes were compared using a caspase-3 cleavage sequence PDEVDR.
Comparison was made pairwise, with MitoNEET, Cytokeratin-8 and Calnexin probes
with EYFP and mGFP (upper row) being compared to an NES probe with mCherry
(lower row). Images show frames at time zero and a frame shortly preceding
apoptotic body formation. Scale bar: 10m.
Supplemental figure 3. a. Comparison of localization probe cleavages in wild-type
HeLa cells. Images show the comparison between Calnexin-LQTDG-YFP and NESLQTDG-mCherry after induction. Plots show the comparison between plasma
membrane, NES and ER probes. Plasma membrane probe is more efficiently
cleaved than NES probe, itself more efficiently cleaved than ER probes. b. Same as
a in the fibrosarcoma cell line HT1080. Images show the comparison between
MitoNEET-LQTDG-YFP and NES-LQTDG-mCherry after induction. Plots show the
comparison between plasma membrane, NES, ER and mitochondrial probes. Plasma
membrane probe is more efficiently cleaved than NES probe, itself more efficiently
cleaved than ER and mitochondrial probes. c. Same comparison focused on plasma
membrane and ER probes for the glioblastoma cell line T98G. d. Same as c for the
adenocarcinoma cell line MDA-MB-231. e. Same as c for the non-cancer cell line
MCF-10A. In all cases, the plasma membrane probe is more efficiently cleaved than
the ER one. Scale bars: 10m.
Supplemental Figure 4. a. Cleavage comparison of NES-LQTDG (red diamond and
green triangle) and Calnexin-LQTDG (blue square and orange cross) with (solid line)
and without (dashed line) caspase-6 overexpression in HeLa cells as in Fig. 5e. b.
Same as a with HeLa-CD95. c. Cleavage comparison of NES-LQTDG (red diamond
and green triangle) and NES-DEVDR (blue square and orange cross) with (solid line)
and without (dashed line) caspase-6 overexpression in HeLa cells as in Fig. 5e. d.
Same as c with HeLa-CD95. e. Western blot timeseries of BID (first row, expected
size 22kd, and 15kD for the cleavage product), PARP (second row, expected size
116kD, and 89kD and 24kD for the cleavage products) and caspase-8 (third row,
expected size 55kD, and 43, 41 and 18kD for the cleavage products) with (right side)
and without (left side) overexpression of caspase-6 in HeLa cells. The vertical arrows
indicate when cells were forming apoptotic bodies. * the 43/41kD bands of caspase-8
are partially hidden by a non-specific band. The ladder was acquired at the same
scale with white light and over-imposed on the blots. f. Same as e with HeLa-CD95.
Note that in both HeLa and HeLa-CD95, the pattern of cleavage of the three proteins
is similar with and without caspase-6 overexpression.
Supplemental figure 5. a. Stable knock-down of caspase-8 in HeLa cells was tested
by Western blot. Quantification of the bands showed that the knock-down reduced
caspase-8 expression to 40% compared to wt HeLa cells. b. Comparison of cell
death at 120 minutes after induction for HeLa stably knocking-down caspase-8
(bright field, upper row) and HeLa wt (bright field, lower row). Time of apoptotic body
formation was estimated from bright field images over time. Note the significant delay
of cell death with HeLa stably knocking-down caspase-8. c. BFP-IRES-caspase-8
(upper row) and BFP-IRES-caspase-8mGFP (lower row) were expressed in HeLa
cells where endogeneous caspase-8 was knocked-down. Caspase-8-mGFP
expression could be visualized by a faint signal in the GFP channel (time zero, GFP
column). Cells expressing this construct were visualized by the EBFP2 fluorescence
(blue cells on bright field images). Plotting of time of apoptotic body formation versus
blue fluorescence for both constructs showed no visible difference. d. NES-LQTDGmCherry cleavage in induced NB7 stably expressing caspase-8-mGFP. The amount
of cleavage is limited compared to the other tested cell lines, and did not allow further
spatio-temporal characterization of caspase-8 activity. e. HeLa-cas8KD expressing
caspase-8-mGFP (first row), incubated with mitotracker-red (second row) and
monitored after induction. Note the dotty pattern (e.g., white arrows) and the absence
of correlation with the mitochondrial pattern in the overlay image (third row). f. MCF-7
expressing caspase-8-mGFP monitored after induction. In addition to the dotty
pattern (e.g. arrow at time 42 min), a different pattern (arrow at 83 min) can be
observed. g. Comparison of caspase-8-mGFP transfected (first row) and nontransfected MCF-7 cells (second row), with the same settings of image acquisition
and same contrast enhancement. Note that the mitochondrial pattern is also
observed in non-transfected MCF-7 cells. h. Caspase-8 activity visualization in
induced MCF-7 cells expressing NES-LQTDG-mCherry. The probe was fully cleaved,
but no apoptotic body formation could be observed. Scale bars: 10m.
Supplemental figure 6. Schematic representation of intensity normalization. Cells coexpressing MitoNEET-LQTDG-mGFP and NES-LQTDG-mCherry were first imaged
as a 3D stack. Images were converted to 32-bit, background was subtracted from
each slice and the stack was projected. The same region (white regions on projected
stacks) was then used to measure intensities in both channels. Time series were
acquired with the same imaging conditions. The nuclear intensity of both channels
was measured using the same region of interest (white region in time series images).
After normalization with the total intensity from the projected stack, both intensities
were shifted using the same multiplying factor determined from the average intensity
in the cytoplasm at time 0 from the NES channel (blue region at time 0, NES).