Supplemental Figure Legends
Supplemental FIG. 1. Analysis of T cell development in 8-10 week old Ikzf1+/+ and Ikzf1Plstc/+ mice.
(A). Representative FACS dot plots showing CD4 v CD8 expression in thymocytes of mice at the
indicated ages and genotype. B. Representative histograms showing the overlay of TCR-b, CD3, CD5
and CD69 expression by Ikzf1+/+ and Ikzf1Plstc/+ DN, DP, CD4+ and CD8+ T cells from mice shown in
A.
Supplemental FIG. 2 Sontani et al Oligoclonal expansion of TCRV expressing cells in the thymus of
Ikzf1Plstc/+ mice. (A). Representative FACS dot plots of thymocytes and bone marrow cells from
Ikzf1+/+ and Ikzf1Plstc/+ mice demonstrating the clonal expansion of (A) TCR-V8.3 and (B) TCRV5.1,5.2 expressing cells at different stages of T cell development but these cells are not detected in
the bone marrow. Both Ikzf1Plstc/+ mice (G17.23 10 weeks old and G16.62 12 weeks old) showed
significant increase in thymic cellularity whereas spleen numbers were equivalent to wild type mice.
Supplemental FIG. 3. An example of a DP leukaemic phenotype observed in Ikzf1Plstc/+thymocytes.
Upper panel shows thymocytes from Ikzf1Plstc/+ mice of 12 week old G19.67 with a DP leukaemic
phenotype. Lower panel shows changes in the expression level of T cell markers in the different
subset of Ikzf1Plstc/+ thymocytes associated with the changes in CD4 and CD8 profile.
Supplemental FIG. 4. An example of a DP leukaemic phenotype observed in Ikzf1Plstc/+splenocytes.
Upper panel shows splenocytes from Ikzf1Plstc/+ mice of 12 week old G16.67 with a CD4 leukemic
phenotype. Lower panel shows changes in the expression level of T cell markers in the different
subset of Ikzf1Plstc/+ splenocytes associated with the changes in CD4 and CD8 profile. Ikzf1Plstc/+ spleen
DP cells were compared with wild-type DP thymocytes.
Supplemental FIG. 5 Sontani et al. DP thymocytes from Ikzf1Plstc/+ and age matched Ikzf1+/+
counterpart were sorted and RNA samples were obtained and synthesised into cDNA. Realtime RTPCR was performed to analyse the changes in the level of expression of genes such as Notch target
genes (A) Hes1 and (B) Dtx1, (C) Notch1, (D) Ikaros, (E) Lfng, (F) pTα, (G) Rag1 and (H) Cd25 in
DP cells of IkPlstc/+ compared to Ikzf1+/+. The endogenous control used was Ubiquitin conjugating
enzyme (UBE). Values shown are relative mRNA expression calculated from the fold expression
value of Ikzf1Plstc/+, obtained using the comparative Ct method, divided by the corresponding value
from Ikzf1+/+ samples of the same age. Statistical significant age-groups of IkPlstc/+ animals are shown
by * Students t-test; p value ≤0.05 .
Supplemental FIG. 6. Sontani et al. HD domain coding mutations in Notch1 exons 26 and 27 in
DP cells from Ikzf1Plstc/+ mice. Underlined residues have been found to be mutated in human T-ALL
samples 10, with identical substitutions in exon 27 found here highlighted in purple. Blue residues
indicate missense mutations found in Ikzf1Plstc/+ mice by Mantha et al 24. Red denote additional human
mutations compiled by Gordon et al. 44 Green residues are those mutated in T-ALL arising in mouse
mutant strains studied by O’Neil et al. 20. Position of the S1 protease cleavage site is shown.
a
normal thymus and normal periphery
b
abnormal thymus, normal size thymus, normal periphery
c
abnormal and enlarged thymus, normal periphery
d
abnormal and enlarged thymus, trace of DP T cells in periphery
e
abnormal and enlarged thymus, enlarged spleen with abnormal T cells.
Supplemental FIG. 7 Sontani et al. Carboxy-terminal domain coding mutations in Notch1 exon 34 in
DP cells from Ikzf1Plstc/+ mice. Grey arrowheads denote sites of insertion/deletion-induced frameshift
mutations in IkL/L leukemias 23. Red arrowheads are sites of insertion/deletion frameshift mutation in
human T-ALL studied by Weng et al 10.