SYK is a target of lymphocyte-derived microparticles in the
induction of apoptosis of human retinoblastoma cells
Apoptosis
Qian Qiu, Chun Yang, Wei Xiong, Houda Tahiri, Mathieu Payeur,
Rosanne Superstein, Anne-Sophie Carret, Patrick Hamel, Benjamin
Ellezam, Bussières Martin, Mark Vezina, Przemyslaw Sapieha,
Guoxiang Liu, Pierre Hardy*
Corresponding Author: *Pierre Hardy, Research Center of CHU
Sainte-Justine, 3175 Côte-Sainte-Catherine, Room 2714, Montréal,
Québec, H3T 1C5, Canada. Phone: 514- 345-4931 (ext. 3656); Fax:
514-345-4801; E-mail: [email protected]
SUPPLEMENTAL CAPTIONS AND FIGURES
Captions
ESM_1 siRNAs-p21 downregulated p21 protein in Rb cells. Rb cells
were transfected with 50 nM of scramble siRNA or siRNAs-p21 for 24
hours. The p21 protein levels were analyzed by Western blot (A) and
their relative expression levels (B) were presented as a percentage of the
control (scrambled siRNA set as 100%). *P < 0.05 vs. CTL.
ESM_2 LMPs induced Rb cell death evaluated by cell counting. 5×
105/well of Y79 cells in 24-well microplate were incubated with control
vehicle or LMPs (20 µg/ml) for 24 hours or 48 hours. At each end-point,
live and dead cells were counted by trypan blue staining on TC20™
Automated Cell Counter (Bio-Rad Laboratories). Data are presented as
the percent of viable cells ± SEM from three independent experiments
for each condition. **P < 0.01, ***P < 0.001 vs. CTL.
ESM_3 The expression of phospho-SYK in Rb cells was not affected by
LMPs treatment. (A) The expression of total SYK protein and
phosphorylated SYK (p-SYK) in Rb cells treated with LMPs (20 µg/ml)
for different time periods was detected by Western blot. (B) The relative
p-SYK/SYK was calculated and presented as the percentage of control
and presented as mean ± SEM.
ESM_4 LMPs contain p53, p21 and cleaved caspase-3 proteins.
Representative Western blot of phospho-SYK, total SYK, p53, p21 and
cleaved caspase-3 proteins in 10 µg and 20 µg of LMPs respectively.
ESM_5 Heat-denatured LMPs didn’t prevent significantly the LMPsinduced cell death. 5×105/well of Y79 cells in 24-well microplate were
incubated with control vehicle, 20 µg/ml of LMPs, or heat-denatured
LMPs (95°C 10 min). After 24 hours’ incubation, live and dead cells
were counted by trypan blue staining on TC20™ Automated Cell
Counter. Values are presented as the percent of viable cells ± SEM
from at least three independent experiments for each condition. **P <
0.01 vs. CTL.
A
Scramble siRNA
siRNAs-p21
p21
β-actin
Relative p21 protein
(% of control)
B
120
100
80
*
60
40
20
0
Control
siRNAs-p21
ESM_1
% viable cells
120
**
100
80
***
60
40
20
0
CTL
LMPs
24h
CTL
LMPs
48h
ESM_2
A
LMPs treatment time (hr)
0
12
24
p-SYK
SYK
β-actin
Relative p-SYK/SYK
(% of control)
B
120
100
80
60
40
20
0
0
12
24
LMPs treatment time (h)
ESM_3
LMPs (µg)
10
20
kDa
p-SYK
72
SYK
72
p53
53
p21
17
Cleaved caspase-3
19
β-actin
42
ESM_4
% viable cells
120
100
**
80
**
60
40
20
0
CTL
LMPs Heated
ESM_5