Supplementary Figure legends
Supplementary Figure S1. Flag-Beclin-1 and Bcl-2 variants colocalise in cells. (A)
HeLa cells on coverslips were transfected with Flag-Beclin-1 (Flag-Bec) or
cotransfected with Flag-Beclin-1 and equal amounts of Bcl-2 variants (mit-Bcl-2, ERBcl-2, wt-Bcl-2). Cells were fixed with 3% paraformaldehyde after 48 h, labeled with
antibodies against Bcl-2 and Flag and imaged using confocal microscopy. In the
merged images, yellow color indicates colocalisation of Flag-Beclin-1 with the Bcl-2
variants. Bar, 20 m. (B) After 48 h, some cells were loaded with 100 nM
MitoTracker Orange™ (MTO) for 15 min before fixation to visualise mitochondria,
or stained with anti-KDEL after fixation to visualise the ER. Note little expression of
Flag-Beclin-1 at the ER in cells coexpressing mit-Bcl-2, and little expression of FlagBeclin-1 at the mitochondria in cells coexpressing ER-Bcl-2.
Supplementary Figure S2. Analysis of mit-Bcl-2 and ER-Bcl-2 colocalisation by
coexpression of GFP- and mRFP-tagged variants. Inserts containing the entire coding
region of mit-Bcl-2 or ER-Bcl-2 were subcloned in-frame into the EcoRI site of
pEGFP-C3 (Clontech) and a self-constructed mRFP-expression vector. The junction
sequences between EGFP or mRFP and mit/ER-Bcl-2 are SDLELKLRIPAASFRGR
or KLRS, respectively. Fluorescent proteins are at the N-termini whereas the targeting
sequences are at the C-termini of Bcl-2. HeLa cells were cotransfected with GFP-mitBcl-2 and RFP-mitBcl-2, GFP-ER-Bcl-2 and RFP-ER-Bcl-2, GFP-ER-Bcl-2 and
RFP-mit-Bcl-2, or GFP-mit-Bcl-2 and RFP-ER-Bcl-2. After 24 h, cells were fixed
and imaged by confocal microscopy using a Leica TCS confocal system. Maximal
projection images are shown, obtained from 25 Z stacks. Plots display intensity
profiles along the lines shown in white. Note complete correspondence between the
two fluorescent proteins when they are attached to the same construct, and the lack of
correspondence between them when they are attached to the different proteins. Bar =
10 µm.
Supplementary Figure S3. Activation of caspase activity, caspase-dependent cell
death, and PARP cleavage by the stimuli used in the main text. (A) The caspaseinhibitor Boc-Asp(O-methyl)-CH2F (BAF, 50 µM) suppresses cell death induced by
UVC irradiation (300 J/m2 5 h), Staurosporine (STS, 0.25 µM, 16 h),
Thapsigargin/Tunicamycin (TT, Tg, 2 µM, Tm 10 µg/ml, 16 h), and TNF and
cycloheximide (TNF 100 ng/ml, CHX 20 µM, 5 h). (B) DEVD-ase activity
measured following each of the treatments shown in (A) compared to untreated
controls. (C) PARP cleavage measured following each of the treatments shown in (A)
compared to untreated controls. Note correspondence between the amount of caspase
activity and PARP cleavage.
Supplementary Figure S4. Confocal microscopy of Atg5 MEFs showing Beclin-1
localisation at mitochondria and ER in the presence of mit-Bcl-2 or ER-Bcl-2,
respectively. MEFs were grown on cover slips and transfected with the indicated
plasmids. Before fixation cells were loaded with MitoTracker Orange (100 nM) for 15
min in order to stain mitochondria and ER was revealed using an antibody against the
KDEL sequence. In the merged images (right panels), yellow color indicates
colocalization of Beclin-1-GFP with mitochondria or ER.
Supplementary Figure S5. Beclin1 fails to alter the protection by Bcl-2 against
3MA- and STS-inducible apoptosis. Cell death (left panel) quantified by determining
the proportion of HeLa cells containing condensed, fragmented and Hoechst-positive
or propidium iodine (PI)-positive nuclei. HeLa cells were transfected with the
indicated plasmids and treated one day later with STS (0.25 M) alone or in the
presence of 3-methyladenine (3MA, 10 mM) for 20 h. Error bars, SEM from at least
three independent experiments. * p<0.05, ANOVA followed by Tueky’s post hoc test.
Immunoblot (right panel) for poly-ADP-ribose polymerase (PARP) from HeLa cells
overexpressing Beclin1 or Bcl-2 variants alone or together and treated with STS (0.25
M) alone or in the presence of 3MA (10 mM) for 20 h. Immunoblots were probed
for PARP; blots for Beclin-1, Bcl-2 and tubulin are not shown. Note the more
selective protective efficacy of mit-Bcl-2 in this treatment regime.