Supplementary Figure S1 Characterization of Ras-induced neoplasms. a,
Representative photographs at comparable scale of thymus, liver and spleen
derived from early and late death group animals; normal organs for
comparison. b, Representative blood smears (Wright’s stain) obtained from
animals of the indicated genotypes in a terminal condition. c, Representative
photomicrographs of neoplastic organ infiltrations in the early death group
(lung tissue) and the late death group (liver tissue) indicating reactivity of
neoplastic cells for the T-cell marker CD3 in the early death group and for the
myeloid marker CD11b in the late death group. d, Representative
immunophenotypes of T-cell lymphomas characterized by anti-CD4-FITC and
anti-CD8-PE two-colour flow cytometry (top). Note the similarly distributed
assignations of individual lymphomas derived from different genotypes to the
three immunophenotypes (bottom; n = 5 per genotype). e, T-cell clonality
analysis by genomic PCR using a multiplex panel of VDJ primers for the T-cell
receptor -chain with distinct bands indicating a rearranged clone (as seen in
all lymphoblastic samples tested) compared to the polyclonal smear obtained
from normal thymus DNA.
Supplementary Figure S2 Representative dual-colour flow cytometric
analyses of the indicated antigens reflecting the haematopoietic differentiation
status in various haematopoietic compartments of Suv39h1+/+ and Suv39h1-/mice of matched age.
Supplementary Figure
S3
Invasiveness,
growth
characteristics and
expression of senescence-related genes in mouse embryo fibroblasts and
Ras-driven lymphomas. a, Immunoblot analysis for p16INK4a of lysates derived
from pre-senescent wild-type, Suv39h1-/- and p53-/- mouse embryo fibroblasts
after retroviral infections with Suv39h1 and oncogenic ras or their respective
vector controls as indicated, and -Tubulin expression as a loading control.
Note that p16INK4a levels increase in response to oncogenic Ras, but remain
largely unaffected by the Suv39h1 status. b, Analysis of p16INK4a (top) and
ARF (bottom) protein expression by immunohistochemistry in representative
lymphomas of the indicated genotypes. c, Suv39h2-RT-PCR, demonstrating
that Suv39h1-null lymphomas may still express Suv39h2 transcripts. d,
Photomicrographs of haematoxylin-and-eosin stained lung sections of the
indicated genotypes from the “early death group”, exhibiting massive
infiltration by a blastic population (6/6 “early” cases tested, while 3/3 “late”
cases tested lacked any blastic infiltration). e, Lymphoma-infiltrated spleen
sections of the indicated genotypes stained for the proliferation marker Ki67
(“ctrl. [late]” reflects a histiocytic sarcoma sample). Quantified from 100 cells
per sample (n = 3 per genotype) as averaged positive cells in percent ±
standard deviation. f, Spontaneous apoptosis in situ visualized by a
fluorescence-based TUNEL assay. Shown are two representative cases per
genotype (left) and an integrated signal quantification (right; n = 4 per
genotype) to indicate level and range of Ras-provoked cell death in the
different genotypes. g, Analysis of p16INK4a and ARF protein levels by
immunoblotting of lysates (top) and by immunocytochemistry on cytospin
preparations (bottom) in Bcl2-expressing Ras-lymphomas of the indicated
genotypes 5 days after ex vivo-treatment with 0.1 µg/ml adriamycin or left
untreated for comparison.
Supplementary
Figure
S4
Characterization
of
TSA/DAC-promoted
lymphoma and leukaemia in Eµ-N-Ras transgenic mice. a, Representative
photomicrographs of a haematoxylin-and-eosin [H.E.] and anti-Ki67 stained
lymphoma-infiltrated (arrow) mediastinal lymph node, indicating the high
proliferative capacity of this malignancy (right). b, Comparison of a TSA/DACpromoted with a Suv39h1-null Ras-driven lymphoma/leukaemia sample by
anti-CD3 staining of leukaemic cells indicating their T-cell origin (left), and by
immunostaining for terminal deoxynucleotidyl transferase (TdT, right),
indicating the less mature nature of the Suv39h1-null lymphoma.
Supplementary Table S1 Clinical and pathological characteristics of terminal
disease conditions in Eµ-N-Ras mice of the indicated genotypes grouped
according to their time-to-death.
Supplementary Table S2 Comprehensive summary of haematological
differentiation measured in nucleated cell populations isolated from the
indicated compartments of Suv39h1+/+ and Suv39h1-/- mice (n = 3 per
genotype; all animals were about four months of age) by flow cytometric
immunophenotyping based on the indicated antibodies. Reactivity reflects
percentage of positive cells as follows: “-“ ≤ 0.5%; “(+/-)” 0.5 - ≤ 1.5%; “(+)” 1.5
- ≤ 5%; “+” 5 - ≤ 15%; “++” 15 - ≤ 45%; “+++” > 45%; “(var)” indicates high
variability reflecting that the standard deviation was larger than half of the
mean.
Supplementary Table S3 Chromosomal aberrations of individual primary control
(n = 2), Suv39h1-null (n = 4), and p53-null Ras-lymphomas (n = 3) assessed by
spectral karyotyping.