Supplementary Materials and methods.
PCR genotyping. Detection of the wild type (+), floxed (fl) and deleted () alleles of CnB1 in
genomic DNA of compound mice and leukemic cells was by PCR using 3 primers: P1, 5'GAC AGC TAT ACA GAG AAA CCC TG-3'; P2, 5'-AGC CTC CAC ATA CAC AGA
TAC-3'; and P3, 5’-CAA TGC AGT CCG CTG TAG TTC-3’. The PCR products were 189
bp (+, primers P1/P2), 286 bp (fl, primers P1/P2) and 168 bp (, primers P2/P3). The Cre
transgene was detected as a 596 bp product using the forward primer 5’-ATC CGA AAA
GAA AAC GTT GA-3’ and reverse primer 5’-ATC CAG GTT ACG GAT ATA GT-3’.
Exogenous CnB1 was detected as a 160 bp product by using the forward primer 5’-GTA TGA
TGT TCC TGA TTA TGC TAG C-3’ and reverse primer 5’-CAT GAA CTC TTC CAC GCT
CA-3’. PCR amplification of the HSP70 gene using the forward primer 5’-GCT GAG AAG
CAC CAG GAT TC-3’ and reverse primer 5’-CGG GGT CTC CTT TTC TGT CT-3’ was
used as control.
Flow cytometry analyses. Surface staining of tNGFR leukemic cells was performed with PEconjugated anti-human CD271 antibody (BD Biosciences). Apoptosis assays were carried out
using PE-conjugated-, FITC-conjugated- or Biotin-conjugated Rabbit Anti-active Caspase-3
antibody/Streptavidin APC-Cy™7 (BD Biosciences). BrdU incorporation assays were
performed with APC-conjugated anti-BrdU antibody (BD Biosciences). Flow cytometry
acquisitions were carried out on a FACSCalibur™ analyzer (BD Biosciences) equipped to
detect 4 fluorescent parameters with the assistance of BD CellQuest Software (BD
Biosciences) and data were analyzed with FlowJo Software (Tree Star). ICN1 leukemic cells
were sorted on a FACSAria™III (BD Biosciences) cell sorter on the basis of GFP or tNGFR
expression or as GFP+dsRed+ or GFP+tNGFR+ populations for retrovirally-transduced cells
with the assistance of BD FACSDiva Software (BD Biosciences).
Retroviral- and lentiviral-mediated gene transfer. The cDNA encoding the CnB1 (PPP3R1)
regulatory subunit of mouse calcineurin was subcloned into MigR1 or MicR1, allowing its coexpression with either GFP or its Cherry spectral variant. The cDNA encoding mouse Bcl2
(ThermoScientific) was subcloned into the MigR1 or MSCV-IRES-DsRed vectors, allowing
Bcl2 co-expression with GFP or its dsRed variant, respectively. Retroviral stocks were
obtained following transfection of the PlatE packaging cell line using the calcium phosphate
coprecipitation method, as described(16) and titrated on NIH3T3 cells. tNGFR+ ICN1 leukemic
cells were spin-infected (1,800 g for 2 h at 30°C) at the same multiplicity of infection (moi =
3) with MigR1/CnB1 or MigR1/Bcl2 retroviral supernatants in the presence of 4 g/ml
polybrene (Sigma-Aldrich). To transduce GFP+ ICN1 leukemic cells, infections were carried
out with MSCV-Bcl2-IRES-DsRed. Following infection, leukemic cells (4x106 cells/mouse)
were immediately infused into syngeneic mice and expanded in vivo for further studies. A
cDNA
encoding
the
CnB1
short
hairpin
RNA
(shRNA)
5’-
CGGAGTCTGTAACTTTATTTAT-3’ (resynthesized from the insert and loop of GIPZ
#V2LHS_131430 from ThermoScientific) was subcloned as a BamH1-Xho1 insert into BglIIand Xho-1-digested pSuper. The H1 promoter-insert was retrived by EcoR1 digestion and
sucloned inti pTRIP/U3-MND-GFP and the orientation of the insert determined by DNA
sequencing. Lentiviral-mediated gene transfer into human T-ALL cells was as described
previously(37), except that transduced leukemic cells were grown for 24 hours on MS5 stromal
cells before sorting as hCD45+GFP+ cells.
Microarray analysis. Total RNA was isolated from flow cytometry sorted leukemic cells
(GFP+ for leukemias ICN1; RCT2; CnB1fl/Δ #204 and #206; tNGFR+ for leukemias ICN1;
RCT2; CnB1fl/fl #3) 5 days following treatment with either Tamoxifen or carrier solvent, using
the Rneasy kit (QIAGEN). cRNA synthesis and hybridization of Mouse GeneChip® 430 2.0
arrays (Affymetrix) were according to the manufacturer’s instructions, as described
(http://www-microarrays.u-strasbg.fr). Raw data were processed with Affymetrix Expression
Console software version 1.1 to calculate probeset signal intensities, using Robust Multi-array
Average (RMA) algorithms with default settings and further analyses and visualization were
made using EASANA® (GenoSplice technology, www.genosplice.com), which is based on
the GenoSplice’s FAST DB® annotations [PMID: 16052034, 17547750]. A paired Student’s
t-test was performed to compare gene intensities in the different biological replicates. Genes
were considered significantly regulated when fold-change was ≥ 1.5 and P value ≤ 0.05. The
distance from the gene signal in a given sample to the corresponding average in all the
samples was calculated for each regulated gene. Corresponding values were displayed and
clusterized with MeV4.6.2 from The Institute of Genome Research using Euclidean distance
and average linkage clustering. Significant KEGG pathways (PMID: 22080510) and GO
terms were retrieved using DAVID (PMID: 19131956) from union of results of all, up- and
down-regulated genes separately. DAVID’s EASE score had to be ≤ 0.1. Briefly, EASE score
is a modified Fisher Exact Test that “penalize” classical Fisher Exact p-value in subtracting
the count of positive agreement by “1”.
Apoptosis and cell proliferation assays. Single cell suspensions of ICN1 T-ALL mouse cells
were prepared from leukemic BM or MS5 co-cultures, fixed, washed and stained with PE
Cleaved Caspase 3 antibody (BD Biosciences; dilution 1/6) when assessing GFP+ leukemic
cells or with the Biotin apoptosis kit/APC Streptavidin antibody (BD Biosciences) for
GFP+dsRed+ leukemic cells, followed by flow cytometry analysis. Human T-ALL cells were
obtained from MS5 co-cultures, labeled with an APC-conjugated CD45 antibody (BD
Biosciences; dilution 1/100) and PE-conjugated Annexin V (BD Biosciences; dilution 1/20),
followed by flow cytometry analysis. For proliferation assays, ICN1 leukemic cells-MS5 cocultures were pulse-labeled for 30 min with 10 M Bromodeoxyuridine (BrdU), leukemic
cells identified by cell surface expression of tNGFR, fixed, stained with APC-conjugated antiBrdU antibody (1/50 dilution) and 7-AAD (1g/ml; BD Biosciences) and analyzed by flow
cytometry analysis. Human T-ALL cells were labeled with an APC-conjugated hCD45
antibody (BD Biosciences; dilution 1/50), fixed, stained with a VE450-conjugated antKi67
antibody (BD Biosciences; dilution 1/10) and analyzed by flow cytometry.
Adhesion, motility and migration assays. For flow adhesion assays, a monolayer of MS5 cells
was prepared in a six channel μ-Slide (m-Slide VI0,4 IbiTreat, Ibidi Gmbh). Flow cytometrysorted leukemic cells (0,5x106 cells) in pre-warmed RPMI 1640 medium containing 15% FBS
were perfused during 1 minute with a shear flow of 0.04 dyne/cm2 with an automated syringe
pump (PHD2000; Harvard Apparatus). Shear flow was subsequently stepwise-increased to 1,
5 and 10 dyne/cm2. Fluorescence images in a 0.157-mm2 microscopic field were recorded
with an Inverted Nikon Eclipse Ti microscope using a 20X objective and an EMCDD Evolve
camera (Princeton Instruments). Images were processed with MetaMorph (Molecular Device)
and adherent cells were counted with ImageJ software. For migration assays, MS5 cells were
seeded on 12-well dishes (BD Biosciences) and cultured 24 hours to obtain a confluent
monolayer. Flow cytometry-sorted leukemic cells (0,5x106 cells) were seeded and co-cultured
with MS5 cells for 18 hours. Phase-contrast images were obtained every 10 sec for 15 min at
37°C using a Leica DMIRBE microscope, a Plan APO 20X/0.35 PH1 objective and coolSnap
cf (photometrics) camera, in a 0.233-mm2 microscopic field using Metamorph software.
Frame-by-frame displacements and velocities of 40 randomly selected leukemic cells
movements were calculated by manually tracking individual cells for 15 min using the ImageJ
software on manual tracking mode. Leukemic cells transmigration was assayed using Boyden
chambers (3-m pore size filters; BD Biosciences) pre-coated with a confluent monolayer of
MS5 cells, or the HUVEC (Clonetics Umbilical Vein Endothelial Cell Systems, Lonza) or
MS1 (ATCC CRL-2279™, kindly provided by Dr. Lecanda, Center for Applied Medical
Research, Pamplona, Spain) endothelial cell lines. A total of 106 leukemic cells were seeded
in 200 µl of migration medium (RPMI 0% FBS) on the upper chambers. CXCL12 (200
ng/ml) in RPMI was added in the lower chamber as chemoattractive agent. Leukemic cells
migration was for 6 hours at 37°C. The number of cells that had migrated into the lower
chamber was determined by flow cytometry.
Western blot and Immunofluoresence analyses. Whole-cell extracts obtained from flow
cytometry-sorted leukemic cells were processed for western blots, using antibodies to
NFATc1 (sc-7294), NFATc2 (sc-7296), ERK2 (sc-154) and SAM-68 (sc-333), all from Santa
Cruz Biotechnology. Antibodies to calcineurin B1 (clone CN-B1) and -Actin (clone AC-15)
were from Sigma-Aldrich. The Bcl2 antibody was from BD Biosciences. For
immunofluorescence analyses, flow cytometry-sorted leukemic cells were fixed immediately
or seeded in 8-wells culture slides (BD Biosciences,) at a density of 5x105 cells per well in
RPMI 15% FBS for 6 hours in the presence or not of a confluent layer of MS5 cells.
Leukemic cells were fixed in acetone/methanol 1:1 for 10 minutes at 4ºC. Non-specific
binding was blocked by incubation in 5% FBS for 30 min. Cells were incubated overnight at
4°C with anti-NFATc2 antibody (1:50; Santa Cruz Biotechnology), followed by anti-mouse
Alexa-Fluor 488 (1:50; Invitrogen, CA, USA; 30 min at room temperature). Nuclei were
stained with Hoechst (1:10 000) for 30 seconds and slides were mounted with ProLong Gold
Antifade (Invitrogen, CA, USA). Negative controls were carried out by omission of the
primary antibody
Homing assays. CnB1fl/Δ and CnB1Δ/Δ GFP+ leukemic cells were flow-cytometry sorted from
the BM of leukemic mice and infused intravenously (5x106 cells/mouse) into lethally
irradiated (8.125 Gy) syngeneic recipient mice. BM, spleen and liver were collected 18 hours
later, fixed in 2% paraformaldehyde for 20 min and analyzed for GFP+ leukemic cells by flow
cytometry.
Supplementary figure legends
Supplementary Figure 1. Calcineurin inactivation induced at low tumor burden delays
T-ALL expansion in vivo. (A) Mice were infused with the ICN1-induced leukemias of the
indicated genotype and treated with either Tam or So (n = 3 per group) at low tumor burden
(0.5-1% leukemic cells in BM). At the indicated time points leukemic burden was examined
in BM and spleen of these mice by flow cytometry. ***P = .0003 and *P = .011 (BM day 5
and day 7, respectively); **P = .0059 and **P = .0015 (spleen day 5 and day 7, respectively).
Supplementary
Figure
2.
Calcineurin
inactivation
does
not
modify
the
immunophenotype of leukemic cells. Mice were infused with the ICN1-induced leukemias
of the indicated genotypes and treated with either Tam or So (n = 3 per group) at high tumor
burden (>10% leukemic cells in BM). GFP-gated leukemic cells (leukemia # 206, #21) or
tNGFR-gated leukemic cells (leukemia #3) obtained from So- (left panels) and Tam- treated
(right panels) mice were analyzed 5 days after the start of treatment for CD4 and CD8
expression by flow cytometry. Data depicted for 1 mouse are representative of the group
analyzed.
Supplementary Figure 3. Analysis of CnB1 deletion in leukemic cells that occasionally
escaped Cre-mediated excision of the CnB1 floxed allele. Leukemic cells obtained from
Tam-treated mice (left panel) as described in Figure 1D and genotyped as CnB1 Δ/Δ before
infusion into recipient hosts occasionally killed recipient mice with delayed latency (e.g.
leukemia #20 in Table 1). Examination of the CnB1 genotype in tumor cells recovered from
these recipients (n = 2) revealed the presence of the floxed (fl) CnB1 allele (right panel). PCR
amplification of HSP70 is used as control.
Supplementary Figure 4. Enforced Bcl2 expression confers an advantage to ICN1
leukemic cells in vivo. (A) ICN1; RCT2; CnB1fl/fl leukemic cells (tNGFR+) transduced with
retroviral vectors carrying either a Bcl2-IRES-GFP expression cassette (Bcl2), or the control
IRES-GFP cassette (Control) were infused into syngeneic recipient mice. Non-transduced
(NT) and GFP-transduced leukemic cells were flow-sorted and analyzed by western blot for
Bcl2 expression. ERK-2 expression is shown as loading control. (B) Leukemic cells of the
indicated genotypes were intravenously infused into syngeneic recipient mice (n = 3) and %
GFP+ cells examined at each round in the BM of terminally-ill animals. The leukemic cells
inoculum used (3X106 leukemic cells/mouse) and the number of cells retrieved from
terminally-ill animals (>500X106 leukemic cells/mouse) represent a mean 7-8 population
doublings. At each transplantation step, an enrichment in BCL2-expressing cells is observed,
implying that enforced BCL2 expression confers an in vivo advantage to Cn-proficient
leukemic cells. **P = .0156 and ***P = .0003. Data are represented as the mean  s.d.
(Student’s t-test).
Supplementary Figure 5. Calcineurin inactivation impairs ICN1 leukemic cell
migration, adhesion, and proliferation. (A) Calcineurin is required for ICN1 leukemic cells
transmigration. Flow-sorted leukemic cells of the indicated genotypes were seeded in the
upper compartment of Boyden chambers (n = 3) containing a confluent layer of either BM
stroma-derived MS5 (right), or mouse endothelial MS1 cells (middle) or HUVEC cells (left).
Leukemic cell directed migration towards CXCL12 or FBS in the lower chamber was
recorded and represented as fold-migration over leukemic cells migrating to medium lacking
chemoattractants. *P = .0369 (control), *P = .0127 and **P = .0092 (HUVEC cells, CXCL12
and FBS, respectively); * P = .0129 and **P = .0041 (MS1 cells, CXCL12 and FBS
respectively) and *P = .0139 and **P = .0014 (MS5 cells, CXCL12 and FBS respectively).
(B) Calcineurin regulates ICN1 T-ALL cells adhesion to MS5 stromal cells. Flow-sorted
leukemic cells of the indicated genotypes were perfused under low shear flow (0.04
dyne/cm2) over MS5 cells. Shear force was subsequently increased stepwise to higher values
and the number of adherent cells recorded by live cell imaging in 10 different fields. ***P 
.0001. (C) Calcineurin regulates ICN1 T-ALL cells proliferation in MS5 co-cultures. Flowsorted leukemic cells of the indicated genotypes were seeded on MS5 as triplicates. %
leukemic cells in S phase was determined 24 h later by BrdU pulse-labeling. ***P = .0002.
Data are represented as mean  s.d. (Student’s t-test).
Supplementary Figure 6. CsA and FK506 effects on ICN1 leukemic cells proliferation.
(A) ICN1 leukemic cells of the indicated genotypes were cultivated in RPMI supplemented
with 20% fetal bovine serum, conditions that allowed expansion of Bcl2-expressing ICN1
cells in absence of MS5 stromal cells. Individual cultures were either kept untreated (NT), or
were treated with increasing amounts of Cysclopsorin A (CsA) and cells counted at days 2
and 3, as indicated. Data are represented as relative fold-proliferation over non-treated cells
and as mean  s.d (n = 3; Student’s t-test). ***P  .0001 (CsA-treated CnB1fl/Δ/Bcl2 cells at
days 2 and 3); ***P = .0010 and ***P < .0001 (CnB1Δ/Δ/Bcl2 cells treated with 1.25 M and
5 M CsA, respectively, analyzed at day 2); ***P = .0007 and ***P < .0001 (CnB1Δ/Δ/Bcl2
cells treated with 1.25 M and 5 M CsA, respectively, analyzed at day 3). Cell proliferation
by BrdU labeling at day 2 (right panels) was also assessed. Data are represented as mean 
s.d. ***P < .0001 (CnB1fl/Δ/Bcl2 and CnB1Δ/Δ/Bcl2 cells treated with 5 M CsA) (comparing
mean percentages of n = 3; Student’s t-test). Note that proliferation of Cn-deficient leukemic
cells is efficiently inhibited by CsA. (B) As in A, except that individual cultures were either
kept untreated, or were treated with increasing amounts of FK506 and cells counted at days 2
and 3. Data are represented as fold proliferation over non-treated cells and as mean  s.d (n =
3; Student’s t-test). **P = .0081, **P = .0014 and **P = .0033 (CnB1fl/Δ/Bcl2 cells treated
with 0.2M, 1.25 M and 5 M FK506, respectively, analyzed at day 2); ***P = .0002
(CnB1fl/Δ treated cells with 1.25 M FK506, analyzed at day 3) and ***P < .0001
(CnB1fl/ΔBcl2 cells treated with 0.2 M and 5 M FK506, respectively, analyzed at day 3).
Cell proliferation was also assessed by BrdU labeling at day 2. Data are represented as mean
 s.d. *P = .0154 and *P = .0336 (CnB1fl/Δ/Bcl2 cells treated with 1.25 M and 5 M FK506,
respectively) (comparing mean percentages of n = 3; Student’s t-test). Note that proliferation
of Cn-deficient leukemic cells is not significantly affected by FK506 treatment.
Supplementary Table legends
Supplementary Table 1. EGIL stages, molecular subtypes and Notch pathway
mutational status of human T-ALL used in this study.
Supplementary Table 2. Calcineurin is required for leukemia-initiating activity of ICN1induced T-ALL. Mice carrying ICN1-induced leukemias of the indicated genotypes were
treated with Tamoxifen or the carrier solvent, as described in Figure 2B. Leukemic cells of
the resulting Cn+ (fl/+; fl/; fl/fl) and Cn-deficient () genotypes were intrafemorally
infused into groups of syngeneic recipient mice, as indicated. When mice infused with Cnproficient leukemic cells became terminally-ill, cohorts were sacrificed and leukemic burden
was analyzed as % GFP+ (or tNGFR+ for leukemia #3) leukemic cells in bone marrow (BM)
and spleen. Where indicated, a subset of the mice infused with Cn-deficient leukemic cells
was maintained for longer periods of time and sacrificed at the indicated time points. NA: not
applicable. Data are represented as mean  s.d. (Student’s t-test).
Supplementary Table 3. Calcineurin is required for leukemia re-initiation from de novo
homed leukemic cells. Syngeneic mice were infused with ICN1 leukemic cells of the
indicated genotypes and treated with either Tamoxifen (Tam) or the carrier solvent (So) 24
hours later, as described in Figure 2C. Cohorts were sacrificed when mice from control
groups (Tam- and So-treated mice infused with CnB1fl/+ leukemias and So-treated mice
infused with CnB1fl/Δ or CnB1fl/fl leukemias) became terminally ill. Where indicated, a subset
of the Tam-treated mice infused with CnB1fl/ or CnB1fl/fl leukemic cells was maintained for
longer periods of time and sacrificed at the indicated time points. Leukemic burden was
analyzed as % GFP+ (or tNGFR+ for leukemia # 3) leukemic cells in bone marrow (BM) and
spleen. NA: not applicable. Data are represented as the mean  s.d.
Supplementary Table 4. CnB1 gene deletion impairs clonogenicity of ICN1-induced
leukemias ex vivo. Leukemic cells of the indicated genotypes were obtained following
treatment of mice carrying either ICN1; RCT2; CnB1fl/+ #25 or ICN1; RCT2; CnB1fl/fl #3
leukemias with either Tamoxifen or carrier solvent at late disease stage. Replicates of 5-fold
serial dilutions of flow-sorted leukemic cells of the indicated genotypes were seeded on
confluent MS5 cells. The number of wells scoring positive for the presence of leukemic cells
was recorded and the leukemic cells cloning efficiency was estimated by Poisson statistics.
Data are representative of 2 independent experiments. A 2 test was used to compare cloning
efficiencies of leukemic cells of the indicated genotypes at different dilutions. The uncertainty
on the number of positive responses has been estimated according to the binomial
distribution.
Supplementary Table 5. Microarray analysis of the expression of genes differentially
deregulated (fold-change ≥ 2.4) in vivo between the Cn+ and Cn-deficient version of
ICN1 leukemias.