Supplementary Information
Materials and methods
Immunoblot assays
The analyses were conducted as previously described.1 Cells were collected and washed with cold
PBS. Cell pellets were lysed with a lysis buffer containing 10 mM Tris HCl (pH 7.5), 1% NP40, 150
mM NaCl, 0.1% SDS, and 1% deoxycholic acid salt. Protease inhibitor cocktail III (Calbiochem, San
Diego, CA) with or without phosphatase inhibitors [1 phosphatase inhibitor cocktail I (Sigma-Aldrich,
St. Louis, MO) plus 1 mM sodium meta-vanadate, 1 mM activated sodium ortho-vanadate, 10 mM
NaF, and 10 mM -glycerol phosphate] were added to the cell lysates. Protein concentrations were
determined using Bradford protein assay reagent (Bio-Rad Laboratories, Hercules, CA). Antibodies
against phosphor-Src (pY416), Src, phosphor-CDK2 (pT160), Cyclin D3 (DCS22), c-Myc, and -actin
were purchased from Cell Signaling Technology, Inc. (Danvers, MA). Antibodies against p-Tyr (pY99),
Lyn (44), Hck (H-85), p-Hck (pY411), Blk (K-23), CDK2 (M2), CDK3 (Y-20), CDK4 (C-22), CDK6 (C21), CDK8 (C-19), Cyclin D2 (C-17), and BCL2 (DC21) were purchased from Santa Cruz
Biotechnology, Inc. (Santa Cruz, CA). Anti-Lyn (pY396) was purchased from Epitomics, Inc.
(Burlingame, CA). Purified XIAP antibody was purchased from BD Biosciences (San Diego, CA).
Immunoprecipitation assay
Cells were lysed as described for Immunoblot assays. Equal amounts of cell lysates (300-600 µg in
lysis buffer) were pre-cleared with protein G agarose (Upstate USA, Inc., Charlottesville, VA) at 4°C
for 1 hour with agitation. The pre-cleared lysates were subsequently incubated with 2-3 µg of anti-Blk
antibody overnight at 4°C and then incubated with 30 µL of the agarose beads for 3 hours at 4°C with
agitation. The beads were washed four times with lysis buffer and separated with a NuPAGE Novex
Bis-Tris Gel (Invitrogen, Carlsbad, CA) and blotted to PVDF membranes (Bio-RAD, Hercules, CA). pBlk was detected with anti-p-Tyr (pY99).
Cell cycle analysis
Cells were treated with 100 nM Dasatinib or DMSO for 24 to 72 hours. Cell cycle was determined
using the FITC BrdU Flow kit (BD Biosciences, San Diego, CA) following the manufacturer’s
instructions. Briefly, cells were labeled with bromodeoxyuridine (BrdU) at a final concentration of 10
μM for 1 hour, fixed, permeabilized, stained with FITC-conjugated anti-BrdU antibody and 7-amino-
actinomycin D (7-AAD) for 20 minutes at room temperature, and 10,000 events were collected and
analyzed by FACS Calibur using FL-1 and FL-3 channels.
Intracellular phosphospecific flow cytometric assays
Intracellular phosphospecific flow cytometric analyses of Syk, BLNK and PLC2 were performed using
antibodies and other reagents from BD BioSciences (San Diego, CA) according to the protocols
provided. Briefly, 2x106 cells were incubated with 200 nM Dasatinib or DMSO in 300 L media for 30
minutes at 37C in the 5% CO2 incubator. For this type of analysis, the optimal concentration of
dasatinib was determined to be 200 nM. Lower concentrations of the drug did not yield consistent
results possibly due to indirect effects of dasatinib on these molecules during short periods of drug
exposure. Following 30-minute dasatinib treatment, cells were stimulated for 10 minutes with 10
g/mL of each of goat F(ab’)2 anti-human IgM and goat F(ab’)2 anti-human IgG (Southernbiotech,
Birmingham, AL).2.3 Subsequently, cells were fixed, permeabilized and labeled with the following
antibodies according to manufacturer’s protocols: PE-conjugated p-Syk (pY348), PE-conjugated pBLNK (pY84) and PE-conjugated p-PLC2 (pY759). Cells were then washed with the stain buffer and
10,000 events were collected and analyzed using the FL-2 channel on FACS Calibur.
References
1
Wang YL, Frauwirth KA, Rangwala SM, Lazar MA, Thompson CB. Thiazolidinedione activation of
peroxisome proliferator-activated receptor gamma can enhance mitochondrial potential and promote
cell survival. J Biol Chem 2002; 277: 31781-31788.
2
Ioannis Anagnostopoulos FD, Harald Stein. Diffuse Large Cell Lymphomas. In: Knowles DM, ed.
Neoplastic Hematopathology (ed 2nd). Lippincott Williams &Wilkins, Philadelphia, PA; 2001:855-913
3
Hans CP, Weisenburger DD, Greiner TC, Gascoyne RD, Delabie J, Ott G et al. Confirmation of the
molecular classification of diffuse large B-cell lymphoma by immunohistochemistry using a tissue
microarray. Blood 2004; 103: 275-282.
Supplementary Table 1. IC50 of tyrosine kinase inhibitors in DLBCL
Cell lines
Cell Lines
Imatinib (M)
PP2 (M)
Dasatinib(nM)
K562
Ly7
Ly8
Ly10
VAL
Ly1
Ly18
Ly3
1.2
20.4
9.7
40.1
14.3
22.9
15.6
24.1
5.4
10.8
3.8
10.7
9
11.9
21.6
470.7
0.2
24.3
15.3
13.7
12.3
105.2
231.9
>800
Light shading indicates sensitivity to the inhibitor and dark shading
indicates resistance to the inhibitor.
Figure Legends
Supplementary Figure 1. Effects of Dasatinib on phosphorylation of CDK2 and apoptotic
proteins. (a) Immunobloting of p-CDK2 at Threonine 160 in indicated DLBCL cell lines. Cells were
collected at 72 hrs following dasatinib treatment at indicated concentrations. (b) Immunoblotting of
BCL2 and XIAP in indicated DLBCL cell lines. Cells were collected at 72 hrs following dasatinib
treatment at indicated concentrations.
Supplementary Figure 2. Effects of Dasatinib on viability of primary DLBCL cells. Viability was
determined by propidium iodide staining followed by flow cytometric analysis. Viability of three cases of
DLBCL
at
various
time
points
with
or
without
dasatinib
at
indicated
concentrations.
a
K562
nM
0
50 100 200
LY10
0
50 100 200
LY7
0
50
100 200
LY18
0
50
100 200
LY3
0
50
100 200
p-CDK2
-actin
b
K562
nM
0
50 100 200
LY10
0
50 100 200
LY7
0
50
100 200
LY18
0
50
100 200
LY3
0
50
100 200
BCL2
XIAP
-actin
Supplementary Figure 1.
Viability (%)
100
80
60
40
20
0
DLBCL5
0
24
48
72
96
100
80
60
40
20
0
DLBCL6
0
24
48
72
Time (hr)
Supplementary Figure 2.
96
100
80
60
40
20
0
DLBCL7
DMSO
Das 100nM
0
24
48
72
96