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Supplementary figure legends
Fig. 1. Stable expression of H-ras and v-src in NRK cells (A) NRK wild type (WT)
and NRK cells that have been stably transfected with an expression vector for
the activated H-ras gene were photographed with phase contrast at a 200-fold
magnification. (B) Extracts (40 g) of wild type and H-ras transfected NRK cells
were probed with an antibody against H-ras (lower panel) or -actin (upper
panel). (C) Morphological appearance of NRK cells transformed by the v-src
oncogene. Phase contrast pictures were taken with a 200-fold magnification. (D)
Total cell extracts (50 g) of the indicated cells were probed with an anti-src
(lower panel) or an anti--actin antibody (upper panel).
Fig. 2. Cellular phenotypes used for apoptosis quantification Top, Shown are
microphotographs of HUVEC cells transfected with GFP (as a control) or the
ORCTL3-ECFP plasmid and stained with Hoechst to assess the morphological
changes of the nucleus during apoptosis. Hela cells are shown in the right-hand
panels as a positive control for ORCTL3-induced apoptosis. The plasmids that
were transfected into these cells are also indicated on top of the panels. Bottom,
WT and H-ras-transformed NRK cells were transfected with the indicated
plasmids and microphotographs taken 24 hours later with a suitable fluorescence
filter.
Fig. 3. Examples of morphologically apoptotic cells and cells with different
degrees of internal accumulation of ORCTL3. Hela cells were transfected with an
ORCTL3-ECFP expression vector using the Effectene transfection reagent
(Qiagen). The arrows point to cells that serve as examples of the three cell
morphologies scored for figure 6A: a prominent intracellular accumulation of the
fusion protein, a more balanced internal/membrane distribution and an apoptosis
morphology as ascertained 24 hours post-transfection using a fluorescent
microscope.
Fig. 4. Comparison of apoptosis induction by tunicamycin in Hela and HUVEC
cells. HUVEC (A) and Hela (B) cells were treated with various tunicamycin
concentrations and apoptosis was determined at the indicated time points by
phenotype quantification (0.288p0.671 at 24h, 0.080p0.859 at 12 h,
0.014p0.052 at 6 h, n=3).