Supplementary Figure Legends
Figure S1. Overexpression of Vmp1 induces apoptosis. a. Effect of Vmp1
overexpression on Caspase-3 activation. HEK293 cells were cultured in 24-well
plates at a density of 32,000 cells/well, and were transfected with YFP tagged cDNAs
at 200ng DNA/well, using Effectene transfection reagent. Mock transfected cells were
used to assess the autofluorescence as well as the background level of Caspase-3
activation; CDK2 (Cyclin dependent kinase2) was considered as negative control, and
CIDE-C (C- terminal domain of Cell Death Inducing DFFA-like Effector) as positive
control. The adherent and floating cells were collected 1, 2 and 3 days after
transfection, stained with Caspase-3 antibody, measured by FACS and analyzed as
described previously (Sauermann et al., 2007) (Hahne et al., 2006). Transfections with
YFP-CDK2 did not result in Caspase-3 activation, CIDE-C induced strong Caspase-3
activation in the transfected cell population from day 1 onwards, whereas Vmp1
induced apoptosis in both transfected and untransfected populations from day 2. b.
Vmp1 induces apoptosis in the neighbouring untransfected cells. HEK 293 cells
overexpressing YFP tagged Vmp1 induce membrane blebbing in the neighbouring
untransfected cells (shown with an arrow in left panel). Staining the cells with Dapi
revealed the presence of nuclear fragmentation, a sign of advanced apoptosis, in the
transfected and neighbouring untransfected cells (shown with arrow in right panel).
Figure S2. Calcium switch assay with MCF-7 cells. MCF-7 cells were seeded and
allowed to attach on cover slips in 6 well plates (1x106 cells/well) in DMEM/F12
medium (Gibco BRL) with 1.05 mM Calcium chloride for 2 hours. Then, the medium
was replaced with calcium free medium and cells were incubated at 37°C overnight.
On the following day, the medium was replaced with medium containing 1.05 mM
calcium. Cells were fixed and stained at RT with specific antibodies against ECadherin (Red) and Vmp1 (Green) at 0hrs (before addition of media with calcium),
and 1hrs, 2hrs and 24hrs after addition of media with calcium. Briefly, for VMP1
staining cells were fixed with 4% PFA for 20 min, washed thrice with PBS and
blocked with 3% BSA for 30 min. Vmp1 antibody was diluted in 3% BSA and cells
were incubated for 30 min. After washing thrice with PBS, cells were permeabilized
1
with 0.2% Triton X-100 and blocked again with 3% BSA. Then the cover slips were
incubated with the E-Cadherin antibody diluted in 3% BSA for 30 min followed by
incubation
with
secondary
antibodies.
E-Cadherin
antibody
(Santa
Cruz
Biotechnology, Santa Cruz, CA) was diluted 1:100 and VMP1 antibody was diluted
1:500 for Immunofluorescence. Alexa 488 and Alexa 568 conjugated antibodies were
used as secondary antibodies (Molecular Probes) at a dilution of 1:500. Fluorescence
images were obtained using a confocal laser scanning microscope with a 63x oil
immersion objective.
Localization of E-Cadherin was partially detected at the plasma membrane in the
0hrs, 1hrs and 2hrs samples. After 24hrs calcium incubation the adherens junction
formation was mostly completed. Vmp1 expression was low or absent in cells grown
in Ca2+ free medium, but it was seen in cells after 1hr of Ca2+ induction and is still
present after 2hrs. In both cases, Vmp1 localized mostly to areas of cells where the
cell junction formation was incomplete. Cells with completely formed junctions
showed low or no expression of Vmp1. The expression level of Vmp1 is reduced after
24hrs of Ca2+ induction.
Figure S3. Calcium switch assay with MCF10A cells. MCF10A cells were seeded
and allowed to attach on cover slips in 6 well plates (7x106 cells per well) in MEM
(Gibco BRL) with 1.8 mM Calcium chloride. The calcium switch assay and antibody
staining was performed as described in Figure S2. Localization of E-Cadherin was
diffuse in the 0hr sample and then localized to the cell junctions in cells treated with
Ca2+. Vmp1 expression was low or absent in cells grown in Ca2+ free medium and
after 1hr of Ca2+ induction. After 2hrs, Vmp1 is expressed in almost all cells and,
similar to the MCF-7 cells, Vmp1 is mostly located to areas without E-cadherin
staining.. After 24hrs Ca2+ treatment, adherens junctions are almost completed and
low or no Vmp1 expression was observed.
Figure S4. Effect of VMP1 downregulation on Caspase-3 activation. a.
Determination of knock down efficiency of BIRC4 siRNA (positive control for
Caspase-3 activation) in HEK293 cells by qRT-PCR. mRNA level of non-transfected
cells was adjusted to 1 and the levels of other transfections (MOCK, GFP and BIRC4)
were calculated in comparison to level of non-transfected cells. ACTB and HPRT1
are housekeeping genes and MOCK are the cells treated only with the transfection
2
reagent without siRNA. b. Morphology of HEK293 cells after transfection with
corresponding siRNAs. After incubation of cells with siRNAs for 48 hours, detached
cells were collected and the attached cells were trypsinized. Morphology of both the
fractions were analysed by FACS according to their forward (FSC-H) and side scatter
(SSC-H) properties. Dashed red gate shows the smaller cells in the left population and
dashed green gate shows cells with normal morphology in the right population. c.
Density plots of Caspase-3 activation. The Caspase-3 assay was performed as
described previously (Sauermann et al., 2007). G represents the cut-off, right side of
which was accepted as activated Caspase-3 signal. In the attached cell population
(upper panel), all transfections resulted in the same level of activation, whereas in the
detached population (lower panel) only positive control (BIRC4 siRNA) showed
activation of Caspase-3.
Figure S5. Phalloidin and Dapi staining of HEK 293 (left panel) and MCF-7 cells
(right panel). After 24-hour incubation of cells with corresponding siRNAs, cells were
stained with Dapi (for nucleus) and phalloidin (for F-actin). In brief, 4% PFA was
given to the cells in the medium and incubated for 15 min at RT. After permeabilizing
with 0.5% Triton X-100/PBS for 5 min, cells were washed with PBS 3 times.
Blocking was done in 3% BSA for 30 min at RT and cells were washed 3 times for 5
min. Phalloidin was diluted (1:40) in 1% BSA and incubated for 30 min. After
washing 2 times with PBS, cover slides were incubated with Dapi, diluted in PBS
(1:1000), for 5 min at dark. Cover slides were washed once with PBS and rinsed with
water and mounted on cover slips. Fluorescence images were obtained using a
confocal laser scanning microscope with a 63x oil immersion objective. No difference
was observed between samples transfected with GFP and VMP1 siRNA in both cell
lines.
References
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Sauermann, M., Hahne, F., Schmidt, C., Majety, M., Rosenfelder, H., Bechtel, S.,
Huber, W., Poustka, A., Arlt, D., and Wiemann, S. (2007). High-throughput flow
3
cytometry-based assay to identify apoptosis-inducing proteins. J Biomol Screen 12,
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