SUPPLEMENTARY INFORMATION
Proteins of the Bcl-2 family do not target the lysosomal compartment during CPT-induced
cell death. Several members of the bcl-2 family undergo translocation to the mitochondria after
a variety of death stimuli, where they finely regulate permeabilization of the mitochondrial
membrane. Similar lysosomal translocation of pro-apoptotic bcl-2-like proteins therefore
represents an attractive hypothesis to further explore the mechanisms of lysosomal rupture. To
date, no experimental evidence supports such involvement of bcl-2 family proteins at lysosomes.
To investigate this hypothesis, a fractionation method of metrizamide-Percoll density gradients
was followed to obtain lysosome- and mitochondria-enriched fractions from control and CPTtreated cells. High -hexosaminidase activity was detected in lysosome-enriched fractions, but
some activity was also detected in mitochondria-enriched fractions indicating lysosome
contamination within mitochondria-enriched fractions (Supplementary Fig. a). Similarly, the
presence of mitochondrial VDAC1 in lysosome-enriched fractions indicated mitochondrial
contamination within the lysosome-enriched extracts (Supplementary Fig. b, c). Thus, the
expression patterns obtained for various bcl-2 family proteins were compared to VDAC1
patterns in lysosome-enriched fractions. In U937 cells, bax, bak, bim-EL and bcl-2 expression
patterns in lysosome-enriched preparations were very similar to that of VDAC1, suggesting that
the bcl-2-like proteins found in lysosome-enriched preparations originated from mitochondrial
contamination (Supplementary Fig. b). Similar results were obtained from lysosome-enriched
fractions of Namalwa cells, where bax, bak, bik, bim-EL and bcl-2 expression patterns correlated
with VDAC1 patterns (with the exception of bim-EL), again suggesting that these proteins
originated from mitochondrial contamination
(Supplementary Fig. c). As a reference, the
expression patterns of the same proteins were monitored in mitochondria-enriched fractions.
Also, the nuclear protein lamin B was absent from both mitochondria- and lysosome-enriched
fractions (Supplementary Fig. c). Even though it is apparently difficult to obtain highly-purified
organelle fractions, our results suggested that bcl-2 family proteins, at least the ones studied, did
not target lysosomes during CPT-induced apoptosis. However, more discriminatory approaches
must be undertaken before reaching a decisive conclusion.
SUPPLEMENTARY MATERIALS AND METHODS
Cellular fractionation. For the isolation of mitochondria and lysosomes, a metrizamide-Percoll
density gradient protocol, modified from Storrie and Madden (Methods Enzymol 1990, 182:203225), was followed. The protocol was described in details elsewhere (Apoptosis 2004; 9:815831). For immunoblot analysis, 20 g of the mitochondria- and lysosome-enriched fractions were
used. The primary antibodies in this study included anti-Porin 31 HL mouse monoclonal
antibody (clone 89-173/045, Calbiochem-Novobiochem Corporation), anti-Bax rabbit polyclonal
antibody (n-20, Santa Cruz Biotechnology), anti-Bak rabbit polyclonal antibody (06-536,
Upstate
Biotechnology),
anti-Bik
rabbit
polyclonal
antibody
(FL-160,
Santa
Cruz
Biotechnology), anti-Bim rabbit polyclonal antibody (202000, Calbiochem), anti-Bcl-2 mouse
monoclonal antibody (100, Santa Cruz Biotechnology), and anti-lamin B mouse monoclonal
antibody (clone 101-B7, Oncogene Research Products). The secondary antibodies were
horseradish peroxidase-conjugated sheep anti-mouse Ig and donkey anti-rabbit Ig (Amersham
Pharmacia Biotech).
-hexosaminidase
activity. For the measurement of -hexosaminidase activity, lysosomal and
mitochondrial-enriched fractions were lysed in 1% Triton X-100 buffer at 4°C for 30 min, then
centrifuged. 20 g-protein aliquots were immediatly assessed for -hexosaminidase activity in a
reaction mixture containing 1.5 mM of 4-methylumbelliferyl N-acetyl--D-glucosaminide in 500
l
of 400 mM acetate buffer (pH 4.4) and 250 mM sucrose. Enzyme activity was monitored
continuously at 37°C in a dual luminescence fluorometer (LS 50B, Perkin Elmer) at excitation
and emission wavelength of 355 nm and 460 nm, respectively. Enzymatic activities were
determined as initial velocities and expressed as relative intensity/min/mg.
SUPPLEMENTARY FIGURE LEGEND
Kinetics of Bcl-2 family protein expression after CPT treatment in lysosome- and
mitochondria-enriched fractions obtained from U937 and Namalwa cells. a) hexosaminidase activity monitored in lysosome- and mitochondria-enriched fractions obtained
from U937 and Namalwa cells. b) Expression patterns of VDAC1, Bax, Bak, Bim-EL and Bcl-2
in lysosome-enriched fractions obtained from control and CPT-treated U937 cells. c means total
cellular extract. A SDS-polyacrylamide gel stained with Coomassie Brillant Blue R-250 dye is
shown as loading control. c) Expression patterns of VDAC1, Bax, Bak, Bik, Bim-EL, Bcl-2 and
nuclear Lamin B in mitochondria- and lysosome-enriched fractions obtained from control and
CPT-treated Namalwa cells. c means total cellular extract. A SDS-polyacrylamide gel stained
with Coomassie Brillant Blue R-250 dye is shown as loading control, and to represent the overall
difference in protein expression patterns between the 2 fractions. All are representative of 3
independent experiments.