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Materials and methods

Cells and reagents


Interleukin-2dependent CTLL-2 cells were cultured as previously reported 6 and
were treated as indicated in the figure legends. B-CLL cells and T cells were obtained
from patients or healthy subjects as described below. Written informed consents were
obtained from all patients, prior to sample collection, according to the Declaration of
Helsinki. The ethic approval for our study was obtained from the local ethic committee
of Regione Veneto on chronic lymphocytic leukemia.
Psora-4, PAP-1, clofazimine, and FITC-labeled anti-kv1.3 antibody were obtained
from Sigma-Aldrich (Milan, Italy). FITC and Alexa568-labeled Annexin V were obtained
from Roche. Tetramethylrhodamine methyl ester (TMRM) and MitoSOX were purchased
from Invitrogen. Q-7BTPI was the generous gift of Prof. C. Paradisi and Dr. A. Mattarei
(Dept. of Chemical Sciences, University of Padua).

B- and T-cell isolation


After obtaining their informed consent, according to the Declaration of Helsinki,
we obtained peripheral blood from 29 untreated patients with B-CLL as confirmed by
clinical, pathologic, and flow cytometric criteria (positive for CD5). Peripheral blood
mononuclear cells (PBMCs) from the patients were isolated by density-gradient
centrifugation

using

the

Ficoll-Hypaque

technique

(Amersham

Biosciences;

Buckinghamshire, UK). The samples were checked for purity by FACS, and the
procedure was repeated if the percentage of cells other than CD19 + B cells exceeded
5%. Non-manipulated peripheral blood B cells were isolated from the PBMCs of healthy
donors by negative selection using the RosetteSep isolation kit for B cells (STEMCELL
Technologies; Vancouver, Canada). Cell type (CD19 +) purity was at least 95%. In some
experiments (n=3), the separation method of sheep red blood cells was used to obtain
distinct populations of B and T cells from the same B-CLL patients.

Western blot analysis


Cells (5 105 for each assay) were lysed with Tris 50mM, EDTA 5mM, glycerol
10%, SDS 2%, and -mercaptoethanol 1% and were marked with pyronin dye (SigmaAldrich; Milan, IT). Proteins from each aliquot were loaded and separated in a 10%
SDS-PAGE gel. The material was transferred onto a polyvinyl difluoride (PVDF)
membrane (Pall Corporation, Pensacola, FLA, USA) and left there overnight at 4C.
Membranes were incubated with various primary antibodies as described in Leanza et
al6. Secondary antibodies (Calbiochem or Santa Cruz Inc.) were conjugated with either
horseradish

peroxidase

or

alkaline

phosphatase

and

were

detected

by

chemiluminescence (Pierce) using film (Kodak) or digital imaging on a Bio-Rad


ChemiDoc XRS apparatus.

Purification of mitochondria from B-CLL cells


Mitochondria were purified as previously described 6. Briefly, cells were
incubated for 30 min on ice in sucrose/TES (300 mM sucrose, 10 mM TES, 0.5 mM
EGTA, pH 7.4). Cells were then lysed in a Dounce homogenizer, and the lysate was
centrifuged at 600 g for 10 min at 4C. The pellet was again processed in the same
way so that recovery could be maximized. The combined supernatants were
centrifuged once at 600 g, and the pellet was discarded. The mitochondriacontaining supernatant from the last step was centrifuged at 6,000 g for 10 min at
4C. The pellet was gently homogenized and suspended in a small volume of TES
buffer. A further purification was obtained by centrifugation (8500 g, 10 min, 4C) on
a discontinuous Percoll gradient (60%, 30%, and 18% Percoll in TES buffer). The
floating material was discarded, and the fraction at the lower interface was collected
and washed three times by centrifugation at 17,000 g for 5 min. The final pellet was
resuspended in TES buffer.

Cell treatment
CTLL-2 cells were treated as previously reported 6. Next, 1 106 B and T cells
either from patients or from healthy subjects were incubated with the various
compounds (as indicated in figure legends) for 24 hs in a 96-well plate in 200 L
DMEM without serum and phenol red. Psora-4 and PAP-1 were used at 20 M unless
otherwise indicated, clofazimine at either 1 or 10 M, as indicated. CSH was used at 4
M, and probenecid at 50 M.

Cell death detection and double staining


After treatment with the various compounds, apoptotic cells were identified by
labeling with FITC-Annexin V at 37C 6. Cells were then collected and analyzed by FACS
(FACS Canto II, BD BioSciences). % of dead cells was determined by the sum of counts
in Q2 and Q4. In double-staining experiments, treated or control cells were incubated
on ice with 0.5 g of FITC-labeled anti-Kv1.3 antibody for 45 min. Alexa-568-Annexin V
was then added, and cells were incubated for 20 min at 37C. Cells were then
analyzed for FITC and Alexa-568 fluorescence by FACS. Human PBMCs, purified with
Ficoll, were treated for 24 hrs with Probenecid + CSH, Probenecid + CSH + PAP-1,
Probenecid + CSH + Psora-4, Probenecid + CSH + Clofazimine. Cells were then
washed, stained with FITC-anti-Kv1.3 and APC-Annexin and analysed by FACS.

Mitochondrial membrane potential and ROS production


Mitochondrial membrane potential was monitored using tetramethylrhodamine
methyl ester (TMRM), and ROS production was followed using MitoSOX. CTLL-2 cells, B
cells, and T cells were incubated for 20 min at 37C with either 20 nM TMRM or 1 M
Mitosox and were washed once. After incubation, the desired compounds were added
(as indicated in the figure legends), and the decrease in TMR fluorescence or the
increase in MitoSOX fluorescence was measured by FACS at various time points over 2
hr6. Some experiments were performed by pretreating cells with either mitochondria-

targeted 0.5 M Q-7BTPI13 or SOD-PEG (60 U/mL) + CAT-PEG (300 U/mL; both from
Sigma) for 30 min at room temperature. In some panels the results are presented as
plots of the median of the fluorescence distribution histograms recorded under the
various conditions and incubation times, normalized by setting the values measured at
time zero as 100%.

Electrophysiological experiments
Whole-cell currents were recorded with an EPC-7 amplifier (List, Darmstadt,
Germany; filter, 1 kHz; sampling rate, 5 kHz) in B cells obtained as described above
and cultured in RPMI for a maximum of 24 hr, as described previously 8. Leak currents
were not subtracted. For whole-cell experiments the bath solution was composed of
150 mM NaCl, 5 mM KCl, 2.5 mM CaCl 2, 1 mM MgCl2, and 10 mM HEPES (pH 7.3). The
intracellular solution contained 134 mM KCl, 1 mM CaCl 2, 2 mM MgCl2, 10 mM EGTA,
and 10 mM HEPES (pH 7.3). Intracellular voltages are reported, and outward currents
are plotted upward.

In vivo experiments in mice


For the in vivo experiments Clofazimine was injected i.p. into mice at 5 g/g
body weight following the protocol described in Leanza et al 6. Blood was taken 24 hr
after injection.