SUPPLEMENTARY MATERIAL
Inhibition of constitutive RAC3 over-expression inhibits H2O2-induced apoptosis
The constitutive over-expression of RAC3 was also analyzed in HEK293 cells. Several
clones were isolated following transfection, and two of them (designated as clone 1 and
clone 2) selected for subsequent experiments. The wild type and RAC3-expressing cells
were exposed to varying concentrations of H2O2 for 24 h, and viability determined
following staining with crystal violet. As shown in Figure 10A, the sensitivity to H2O2 induced cell death was significantly lower than that observed for wild type cells. Similar
results were obtained with the clone 2 (data not shown). Consistent with these results,
transient transfection of one of these clones (clone 2) with a siRNA expression vector
significantly increased the sensitivity of cells to H2O2 -induced cell death (Figure 10B).
RAC3 is physically associated with AIF in T47D cells
Because the RAC3 coactivator is typically over-expressed in breast tumor cells we
performed experiments in order to determine the probably anti-apoptotic role of this
coactivator trough the physical interaction between RAC3 and AIF in the T47D cell
line. We first analyzed the sensitivity of this cell line to several doses of H2O2. The
apoptotic morphology was observed only with 5mM of H2O2 or higher and after 8 h of
treatment (Figure 11) in a few number of cells with a percent of cell surviving of 70 +
10 % SD (three independent experiments) as determined by the Apoptosis assay of
Biocolor kit, being resistant to doses that induce cell dead in HEK293.
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We then analyzed the possible interaction between AIF and RAC3 in order to determine
if this could be similar to that observed in HEK293 over-expressing the coactivator by
transfection, with a co-localization of both molecules and a minimal AIF nuclear
localization after H2O2 stimulation.
Our experiments of confocal microscopy shows that in these cells RAC3 is
cytoplasmatic and nuclear in basal conditions, although some nuclear localization could
also be observed (Figure 11). This is in agreement with the previously reported shuttling
of this coactivator depending of several phosphorylations (Wu et al., 2002; Yan et al.,
2006).
Our experiments show that both molecules are able to physically interact in the
cytoplasm of T47D cells once AIF was released from the mitochondria after 8 h and 16
h, when most of the cell population is not apoptotic. However, in a few cells that show
to be apoptotic a clear AIF and RAC3 nuclear localization is shown, although not colocalization of these molecules is observed at nuclear level.
Therefore, although it has been previously shown that RAC3 plays an important role in
ER-induced breast tumor cell proliferation (Cavarretta et al., 2002; List et al., 2001) the
RAC3/AIF interaction also occurs in these cells, in a similar way that is observed in
cells over-expressing the coactivator by transfection and could represent one of the
mechanisms by which other tumor types that over-express RAC3 and are not dependent
of steroids (Sakakura et al., 2000), are less sensitive to apoptosis, contributing to this
pathological condition.
SUPPLEMENTARY MATERIALS AND METHODS
Cells
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Human breast tumor T47D cells were grown in DMEM supplemented with 5g/ml of
Insulin, 10 % FCS, penicillin (100 units/ml) and streptomycin (100 mg/ml). Cells were
maintained at 37 °C in a humidified atmosphere with 5 % CO2.
FIGURE LEGENDS
FIGURE 10
Cells expressing RAC3 coactivator in a stable and constitutive manner are less sensitive
than wild type cells to hydrogen peroxide-induced cell death.
(A) HEK293 wild type cells or the RAC3 clone 1 were stimulated as indicated by 24 h.
(B) The diagram bars corresponds to the effect of siRNA on surviving in the RAC3
clone 2. Cells were transiently transfected with siRNA or an empty vector and after 24 h
stimulated with 2 mM of H2O2 by another 24 h. *p < 0.001 at least for the lowest H2O2
dose respect to the wild type cells; **p < 0.001 respect to the cells without siRNA
(Tukey's test).
In both cases, surviving was determined by crystal violet staining as in figure 1F and
each value corresponds to the average of triplicates + SD.
FIGURE 11
RAC3 co-localizes with AIF in T47D cells.
Confocal microscopy showing the co-localization of AIF with RAC3 in cells stimulated
with 5 M of H2O2 by 4 or 8 h. In addition to the not-apoptotic cell, one apoptotic cell
where AIF is expected to be found in the nucleus is shown.
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