Legends to Supplementary figures
Figure 1 Induction of mitochondria clustering and nuclear condensation due to IPAS
expression in HEK293T cells. (a and b) HEK293T cells were transfected with
pEGFP-IPAS constructs, stained with MitoRed and Hoechst 33342 and observed using
a fluorescence microscope. Two representative images of cells expressing IPAS WT, C
and C2 were shown in (a). Arrowheads indicate cells with mitochondria localization of
IPAS and mitochondria clustering. A minimum of 100 transfected cells with or without
nuclear condensation per sample was counted and the result is shown in (b). Data
shown in the bar graph are averages ± s.d. of three independent experiments.*, p<0.05
for indicated comparison. **, p<0.01 for indicated comparison.
Figure 2 Subcellular localization of IPAS and its deletion mutants in HEK293T cells
and CHO-K1 cells. (a) HEK293T cells were transfected with pEGFP-IPAS constructs,
incubated with Z-VAD-FMK and stained with MitoRed. Cells with different localization
of IPAS mutants were counted. Data shown in bar graphs are averages ± s.d. of three
independent experiments. N; Nucleus, C; cytoplasm, Mito; mitochondria. (b) HEK293T
cells were transfected with pEGFP-IPAS constructs, incubated with Z-VAD-FMK,
stained with MitoRed or PI and observed using a confocal fluorescence microscope. (c)
Subcellular localization of IPAS in CHO-K1 cells. CHO-K1 cells were transfected with
pEGFP-IPAS, stained with MitoRed and observed using a fluorescence microscope
(top) or a confocal fluorescence microscope (bottom).
Figure 3 Induction of caspase-3 activation by IPAS WT, IPAS C and IPAS C2.
HEK293T cells were transfected with pEGFP-IPAS constructs, stained with an
anti-active caspase-3 antibody and Hoechst 33342 and observed using a fluorescence
microscope. A minimum of 100 transfected cells with or without active caspase-3
staining per sample was counted and the result is shown below. Data shown in bar
graphs are averages ± s.d. of three independent experiments. **, p<0.01 for indicated
comparison.
Figure 4 Binding of IPAS to pro-survival Bcl-2 family members. (a) Binding of IPAS
to Bcl-xL but not to Bcl-2 and Bax in the presence of Z-VAD-FMK. HEK293T cells
were transfected with indicated combinations of expression plasmids, incubated with
Z-VAD-FMK and analyzed by immunoprecipitation with antibody against FLAG
followed by immunoblotting with antibody against Myc. (b) Binding of IPAS to highly
expressed Bcl-2. HEK293T cells were transfected with indicated combinations of
expression plasmids using Lipofectamine 2000 and interaction between IPAS and Bcl-2
or Bcl-xL was analyzed by immunoprecipitation with an antibody against FLAG
followed by immunoblotting with an antibody against Myc. (c) FLIM-FRET analysis of
the interaction between IPAS-Cerulean (IC) and Citrine-Bcl-xL (YB) in living CHO-K1
cells. CHO-K1 cells were transfected with indicated combinations of expression
plasmids. The fluorescence decay curve of Cerulean (shown in blue) represents an
average of fluorescence decay data obtained from the cytoplasmic area of the observed
cells. The decay curve of separately expressed IPAS-Cerulean (shown in black) was also
shown. Data shown are representative of three independent experiments (top). FLIM
images in the presence (IC + YB) or absence (IC) of Citrine-Bcl-xL were shown
(middle). In the FLIM map, color corresponds to the fluorescence lifetime indicated by
a false color scale (middle). Fluorescence decay data were obtained for IPAS-Cerulean
in the presence or absence of Citrine-Bcl-xL in living CHO-K1 cells (bottom). a1 and a2
are the exponential coefficients for the 1 and 2 decay times, respectively. n: number of
cells examined. (d) FLIM-FRET analysis of the interaction between Cerulean-Bcl-xL
(CB) and Citrine-IPAS (YI) in living CHO-K1 cells. Assays were performed as in (c).
Figure 5 Binding and colocalization of IPAS with HIF-1. (a) Binding of IPAS and its
deletion mutants to HIF-1 in HEK293T cells. HEK293T cells were transfected with
indicated combinations of expression plasmids and interaction was analyzed by
immunoprecipitation with an antibody against FLAG followed by immunoblotting with
an antibody against Myc. (b) Subcellular localization of IPAS mutants and HIF-1 in
HEK293T cells. HEK293T cells were cotransfected with pCerulean-IPAS constructs
and a pCitrine-HIF-1 plasmid, stained with Hoechst 33342 and observed using a
fluorescence microscope.
Figure 6 Binding and colocalization of IPAS with HLF. (a) Binding of IPAS and its
deletion mutants to HLF in HEK293T cells. HEK293T cells were transfected with
indicated combinations of expression plasmids and interaction was analyzed by
immunoprecipitation with an antibody against FLAG followed by immunoblotting with
an antibody against Myc. (b) Subcellular localization of IPAS mutants and HLF in
HEK293T cells. HEK293T cells were cotransfected with pCerulean-IPAS constructs
and a pCitrine-HLF plasmid, stained with Hoechst 33342 and observed using a
fluorescence microscope.
Figure 7 Preferential binding of IPAS C to Bcl-xL. HEK293T cells were transfected
with indicated combinations of expression plasmids and interactions of IPAS WT with
Bcl-xL and with HIF-1/HLF were analyzed by immunoprecipitation with an antibody
against Myc followed by immunoblotting with an antibody against FLAG. Interactions
of IPAS C with Bcl-xL and with HIF-1/HLF were similarly analyzed. ns.; non specific
band.