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low high
GSK3
P(S9)GSK3
CD38-APCCD45-Pacific Blue CD123-PEFSC
CD
34
-PE
-Cy7
CD
34
-PE
-Cy7
SS
C
0.3%
A
B
C
50 250200150100(X1000)
50
250
200
150
100
(X1000)
SS
C
50
250
200
150
100
(X1000)
102 105104103 102 105104103
102
103
104
105
102 105104103
102
103
104
105
P(S9) GSK3GSK3
Adh.Sus.
102 105104103
102 105104103 102 105104103
102 105104103102 105104103
102 105104103
FITC
FITC FITC
FITCFITC
FITC
expression
Pre-immune serum
Isotype control
coun
ts40
00
coun
ts25
00
coun
ts 700
0
coun
ts40
00
coun
ts25
00
coun
ts 125
0
low highphosphorylation
Supplementary Figure 1: Representative gating schemes and profiles obtained by flow cytometry of
immature cell populations and of their P(S9)GSK3 and total GSK3content. A- Immature cell
populations (leukemic CD34+38-123+45lo cells and normal CD34+38-45lo cells) were analyzed by flow
cytometry. From each bone marrow sample, total mononuclear (leukemic bulk or normal CD45 lo cells) and
immature cell populations were analyzed. B- P(S9)GSK3 and total GSK3 content of immature cells were
analyzed by flow cytometry and the MFI ratio was calculated. C- P(S9)GSK3 and total GSK3 were
detected by western blotting with the same antibodies used for flow cytometry.
CD
34
-PE
-Cy7
CD
34
-PE
-Cy7
CD38-APC
CD38-APC
CD38-APC
Complex karyotype
Normal karyotype
Normal(healthy donors)
AML
Sus. Adh.
CD38-APC
1,4% 1,5%
15% 14%
0,6% 0,4%
CD38-APC
CD38-APC
CD
34
-PE
-Cy7
CD
34
-PE
-Cy7
CD
34
-PE
-Cy7
CD
34
-PE
-Cy7
100
50
0
BulkAdherent cells
%
AML normal karyotype
Healthy donorsAML complex karyotype
A B
102 105104103
102 105104103
102 105104103
102 105104103
102 105104103
102 105104103
102
105
104
103
102
105
104
103
102
105
104
103
102
105
104
103
102
105
104
103
102
105
104
103
Supplementary Figure 2: Maintenance of immature cell fraction (CD34+38-) upon cell adhesion.
Cells from healthy donors and AML patients (with normal or complex karyotype) were incubated for 1h
in the absence of serum and then allowed to adhere on fibronectin matrix as described in Material and
Methods. After 1h adhesion, the percentage of adhered cells in total mononuclear fraction was quantified
by crystal violet (B, n=3, mean ± S.E.M.). Also, percentages of CD34+38- cells were analyzed before and
after cell adhesion by flow cytometry (A). Data are representative from 16 AML patients with normal
karyotype, 8 AML patients with complex karyotype and 12 healthy donors.
Non-treated control
SB216763
25%
AML CD34+38-123+ cellsMale patient
AML CD34+38-123+ cellsFemale patient
coun
ts60
00
APO2.7-PE-Cy5102 105104103
suspension
coun
ts30
00
APO2.7-PE-Cy5102 105104103
adhesion
32%
coun
ts30
00
APO2.7-PE-Cy5105104103
30%
102
70%
coun
ts50
00
APO2.7-PE-Cy5102 105104103
12% 15%
17%12%
coun
ts30
0
APO2.7-PE-Cy5102 105104103
coun
ts30
0
APO2.7-PE-Cy5102 105104103
APO2.7-PE-Cy5102 105104103
APO2.7-PE-Cy5102 105104103
0
0co
unts
250
0
coun
ts25
00
suspension adhesion
Supplementary Figure 3: Representative P(S9)GSK3, P(S641)GS and APO2.7 flow
cytometry profiles of AML CD34+38-123+ cells in suspension or adhesion. Labelling by
antibodies directed to P(S9)GSK3 and P(S641)GS (glycogen synthase) (a) and by APO2.7-PE-
Cy5 (b) are described in Material and Methods. In a, 2500 cells were counted in average and in b,
apoptotic responses to the GSK3 inhibitor SB216763 are shown.
a-
b-
P(S9)GSK3
P(S641)GS
Sus. Adh.
Supplementary Figure 4: GSK3 modulation and disparity in apoptotic response of immature leukemic cells from male AML patients. After 1h serum starvation and then 1h adhesion on fibronectin, AML samples were treated or not (Ct.) with LY294002 (10 M), Wortmannin (0.1 M), Ara-C (1 M), Etoposide (1 M), NAC (N-acetyl-l-cysteine from Sigma Aldrich, 50 M) or DPI (Diphenyliddonium chloride from Flüka, 25 M). At the end of 1h incubation period, cells were washed, treated for P(S9) GSK3 analysis (a) or incubated in serum-containing medium for 24h for further analysis of P(S9) GSK3(a) andapoptosis (b). P(S9) GSK3 and apoptosis were measured by flow cytometry in immature leukemic fraction (CD34+38-123+ cells) as described in Material and Methods. Results were evaluated as the percentage of apoptotic cells in suspension or in adhesion for each treament. Data are from 16 male AML patients: 12 developed CAM-DR to etoposide (left panel) and 4 to Ara-C displaying also pro-apoptotic response to anti-oxidants NAC and DPI (right panel). Comparison to respective control in suspension or in adhesion, or as indicated: mean ± S.E.M. *P<0.05 **P<0.01 ***P<0.001.
a-
SuspensionAdhesion
Ct. NAC DPI Ara-C Etop.
AML CD34+38-123+ cellsMale patients
70
60
40
30
50
Apoptosis%
20
0
*******
**
*
Ct. NAC DPI Ara-C Etop.
AML CD34+38-123+ cellsMale patients
70
60
40
30
50
Apoptosis%
20
0
*
*
***
b-
102 105104103
FITC
coun
ts0
90
102 105104103
FITCco
unts
09
0102 105104103
FITC
coun
ts0
90
coun
ts
102 105104103
FITC
09
0
Sus. Adh. Adh. + LY294002 Adh. + WortmanninP(S9)GSK3
AML CD34+38-123+ cellsMale patients
LY2940023
1
0
Inhibited GSK3P(S9)GSK3/GSK3
2
1h 24h
Adhesion (n=3) wortmannin
control
MFI ratio
****
***
SuspensionAdhesion
AML CD34+38-123+ cellsFemale patients
Ct.
70
60
40
30
50
Apoptosis%
20
0IGF-1 Testosterone Estradiol
AML CD34+38-123+ cellsMale patients
Ct.
70
60
40
30
50
Apoptosis%
20
0IGF-1 Testosterone Estradiol
Supplementary Figure 5: Hormonal treatment does not modify survival of immature leukemic
cells from female and male AML patients. Serum-starved adherent cells from 4 female and 4 male
AML patients were treated or not (Ct.) with IGF-1 (RD, 3x10-9M), 19-nortestosterone 17-decanoate
(Sigma Aldrich, 10-8M) or 17 estradiol (Sigma Aldrich, 10-8M) for 1h. At the end of the incubation
period, cells were washed and incubated in serum-containing medium for 24h. Measurement of
apoptosis was performed by flow cytometry in immature leukemic fraction (CD34+38-123+
progenitors) using labeling by APO2.7 as described in Materials and Methods. Results were
evaluated as the percentage (mean ± S.E.M.) of apoptotic cells in suspension or in adhesion for each
treatment.
GSK3High level
GSK3Low level P-value
Median age (years)
Gender (%)MaleFemale
CD34+ % (n)
Cytogenetic %nFavorableIntermediateUnfavorable
FAB subtypes (%) M0M1M2M3M4M5
FLT3-ITD % (n)
WBC (x109/L)
Patients % (n) 20 (15) 80 (58)
5447
62533847
070
64147
613342
2513
7262
ns
ns
61 (48)39 (14) <0.05
23 (48)29 (14)
20795114
4703321
Supplementary Table 1: Initial clinico-biological characteristics of 73 AML patients according to
GSK3 expression. The level of GSK3 was analyzed by western blot and groups « high » and
« low » levels were determined by comparison with CD34+ normal cells. FAB, French American
British classification; WBC, White Blood Cell count; CD34+, percentage of CD34+ in the total
mononuclear cell fraction; FLT3-ITD, FLT3 Internal Tandem Duplication; ns, not significant. Patient
characteristics and clinical parameters were compared between different groups of AML patients (high
vs low GSK3 expression and female vs male) using the 2 test.
ns
ns
Supplementary Table 2: Expression of several hormone and adhesion receptors and signaling
molecules in immature leukemic cells from female and male AML patients. Cells from AML patients
were incubated for 1h in the absence of serum and then allowed to adhere on a fibronectin matrix. After 1h
adhesion, protein expression was quantified by flow cytometry in immature fractions (CD34+38-123+ cells) as
described in Material and Methods. Results are expressed as the mean fluorescence intensity (MFI) of stained
samples. Data are from 3 to 9 female and 4 to 16 male AML patients (mean ± S.E.M, ° comparison between
female and male patients P<0.05, *comparison adhesion vs suspension P<0.05). Source of material: Er and
IGF1R polyclonal antibodies (Abcam), 4 monoclonal antibody (Chemicon), Akt and p21 polyclonal
antibodies (Santa Cruz Biotechnology), P(S473)Akt polyclonal antibody (Cell Signaling technology).
suspension adhesion suspension adhesion
ER 3037 ± 247 3381 ± 222 3582 ± 317 4037 ± 420 IGF1R 6683 ± 817 4554 ± 944 6338 ± 1154 5194 ± 918 Integrin 4 20153 ± 4758° 16259 ± 3074 10425 ± 2454 10430 ± 2550 Survivin 2942 ± 344 4088 ± 399 4111 ± 954 3537 ± 616 P21 4782 ± 205 7790 ± 1045* 4554 ± 847 4309 ± 886
Female Male
P(S473)Akt/ Akt 0.29 ± 0.10 0.21 ± 0.08° 1.18 ± 0.20 0.90 ± 0.25 Akt 9494 ± 1581 12915 ± 3281 3892 ± 226 4133 ± 632 P(S473)Akt 2837 ± 300 2790 ± 276 4625 ± 1056 3882 ± 203 P27 5936 ± 802 5347 ± 880 5581 ± 567 4646 ± 588
AML CD34+38-123+ cells
Supplementary Table 3: Discordant sex-related GSK3 phenotypes of one female and one male AML
patients, and U937. Measurement of GSK3 phosphorylation, apoptosis and RACK1 level were performed
as described in Material and methods. Note that AML patients #18 and #28 have no specific clinico-
biological characteristics (not shown).
suspension adhesion suspension adhesion
Male AML patient #28 5.00 2.34 11622 15800
Female AML patient #18 1.21 1.76 2300 1600
U937 2.50 1.90 4000 8000
P(S9)GSK3/GSK3 RACK1 level
AML CD34+38-123+ cells
% apoptosis in adhesion
Cont. Etop. SB21676315 13 35 28 60 27 36 25 60
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