Supplementary Figure Legends
Figure S1.
Increased stability of SETBP1-MT due to decreased ubiquitination (A) Expression of wild-type and
mutant (D868N) SETBP1 in 293T cells transiently transfected with a SETBP1-WT-3×TY1,
SETBP1-D868N-3×TY1, or empty vector (pMYs-IG). In the right 3 lanes, 10µM of MG-132, a
proteasome inhibitor, was added for 12 hours before lysis to prevent degradation in the proteasome.
(B) Impact of wild-type or D868N mutant of SETBP1 on ubiquitination in vitro. HEK293T cells
were
transiently
transfected
with
pcDNA3-HA-Ub-WT,
SETBP1-WT-3×TY1,
SETBP1-D868N-3×TY1, or an empty vector (pMYs-IG) as indicated, followed by IP of TY1
epitope and Western blotting for HA. Cell lysates were also subject to immunoblotting with
anti-TY1 Ab.
Figure S2.
Expression of SETBP1-MT impairs apoptosis and hematopoietic differentiation. (A-B) Relative
expression levels of ASXL1 (A) and SETBP1 (B) mutants of 6 patterns of 32Dcl3 cell lines evaluated
by qRT-PCR using primers common to both the human and the mice sequence. (C) Relative numbers
of viable 32Dcl3 cells transduced with indicated cDNAs using pMYs-IRES-Puro and
pMYs-IRES-GFP vectors following 24 hours of IL-3 withdrawal (mean ± SEM). (D) Cell cycle
distribution of 32Dcl3 cells transduced with indicated cDNAs determined by PI-staining in the
presence
of
1ng/ml
IL-3
(mean
±
SEM).
pMYs-IRES-puro(IP)/pMYs-IG,
pMYs-IP/pMYs-IG-SETBP1-WT,
(E-F)
HL60
cells
transduced
with
pMYs-IP-ASXL1-MT/pMYs-IG,
pMYs-IP/pMYs-IG-SETBP1-D868N,
pMYs-IP-ASXL1-MT/pMYs-IG-SETBP1-WT,
or
pMYs-IP-ASXL1-MT/pMYs-IG-SETBP1-D868N were maintained in RPMI-1640 supplemented
with 10% FBS. The transduced HL60 cells were stimulated with 10-6M ATRA for 3 days. Cytospin
preparations of these cells assessed by Wright-Giemsa staining are shown (E). Proportions of mature
(segmented) cells (%, top) and immature (blastic) cells (%, middle) or the mean fluorescent intensity
(MFI) of CD11b evaluated using FACS analysis (bottom) are shown (F). All data with error bars
indicate the mean ± SEM from duplicated experiments.
Figure S3.
SETBP1-MT transforms ASXL1-MT cells to induce AML in vivo. (A-B) Relative expression levels
of ASXL1 (A) and SETBP1 (B) mutants in the BM cells of control mice (n=2) and AML mice (n=2)
evaluated by qRT-PCR using using primers common to both the human and the mice sequence. (C)
The relative growth rate of BM cells from mice with control (Mock), MDS (ASXL1-MT), and AML
(ASXL1-MT+SETBP1-D868N) in RPMI-1640 supplemented with 10% FBS and 1 ng/ml IL-3.
(D) Cytospin preparations of BM cells of the AML mice cultured in above conditions for 4 weeks
stained by Wright-Giemsa staining. A representative photograph is shown. Original magnification,
×400.(E) Flow cytometric analyses of BM cells derived from the AML mice cultured in vitro for 4
weeks.
Figure S4.
Therapeutic effects of FTY720 on AML cells and the possibility of direct induction of HoxA9/10
locus by SETBP1. (A) Cell lysates from in vitro cultured BM cells and c-Kit+ BM cells (in vivo) of
AML mice were subject to immunoblotting with anti-phopho-PP2A (Y307)Ab, anti-PP2A Ab, or
anti-tublin Ab (in vitro; 1.0μM for 24 hours, in vivo; 10 mg/kg/day, i.p. for 5 consecutive days) (B)
Kaplan-Meier analysis for the survival sublethally irradiated recipient mice transplanted with bone
marrow from primary SETBP1-MT/ASXL1-MT AML mice followed by treatment with vehicle or
FTY720 treatment (n=8, PBS, n=7, FTY 720, respectively). P value was calculated using a log-rank
test.
(C-D) Quantitative ChIP analysis of c-Kit+ BM cells of the control mice and the AML mice
(ASXL1-MT/SETBP1-D868N) using anti-TY1 antibody for the binding of SETBP1 mutant protein.
PCR primers specific to the indicated regions (1-4) of Hoxa9 (C) or Hoxa10 locus (D) were shown.
Results are representative of 3 independent experiments. Transcriptional start sites are indicated as
arrows.
Figure S5.
Gene set enrichment analysis (GSEA) determining specific gene sets or pathways that are positively
or negatively regulated by additional expression of SETBP1-D868N. Comparison of gene expression
from MDS mice expressing ASXL1-MT alone (right side) or AML mice expressing ASXL1-MT and
SETBP1-D868N (left side) revealed that AML mice exhibited positive correlation with gene sets
upregulated in early or intermediate progenitors (A-B), and negatively correlated with those
downregulated in leukemic stem cells (C). Gene expression in AML was inversely associated with
genes enriched in TGF-β1 signaling (D) and targets of SMAD2/3 (E), and positively associated with
those of Myc targets (F). Compared with control mice, MDS mice expressing ASXL1-MT alone
showed correlation with TGF-β1 signaling. The normalized enrichment scores (NES) and P-values
are given.