BEAS-2B
H460
a
#1
#2 #3
#4 #5 Patient number
Sq
Ad Ad
Ad Ad Squamous/Adeno
N T N T N T N T N T Normal/Tumour
b
Fas Expression
100
Receptor-specific
fluorescence
DR5
DR4
β-Actin
#11 #12 #13 #14 #15 Patient number
Sq Ad
Sq
Sq
Ad Squamous/Adeno
N T N T N T N T N T Normal/Tumour
75
50
25
0
H460 A549 H157 H727 34LU BEAS-2B
DR5
DR4
β-Actin
c
Caspase 8 gene expression
d
Caspase 8 activity
8000
1.50
7000
1.25
6000
*
BEAS2B
H460
***
***
5000
1.00
RLU
CASP8/18S
1.75
0.75
4000
3000
0.50
2000
0.25
1000
0
0.00
H460
A549
H157
H727
34LU BEAS2B
Control
SC
FT
24h
SC
FT
48h
TRAIL
6h
Supplementary Figure 1. Caspase 8 expression and activation and Fas expression in NSCLC and normal lung cell lines.
A Western blot analysis of DR4 and DR5 expression in matched NSCLC (T) and normal (N) tissues. Expression was compared to that in H460 and BEAS-2B cell lines
and equal loading was assessed by analysing β-Actin expression. B Cell surface expression of Fas in NSCLC and non-cancer cell lines. Expression was compared to an
isotype control antibody. C Ratio of constitutive caspase 8 gene expression to 18S rRNA expression in NSCLC panel as assessed by quantitative PCR. D Caspase 8
activity in H460 and BEAS-2B cells transfected with 10nM control (SC) or FLIP-targeted (FT) siRNAs for 24 and 48 hours or treated with 20ng/mL rTRAIL for 6 hours.
Caspase-8 activity was shown to be significantly higher in the H460 cells compared to the BEAS-2B cells following FLIP silencing or TRAIL treatment (* denotes p<0.05,
*** denotes p<0.001 as determined by Student’s t-test).
a
b
c
H460
H460
DR4
30
β-Actin
20
10
DR5
0
β-Actin
SC siRNA SC siRNA SC siRNA
Fas
d
DR5
e
15
10
f
H460
H460
50
SC
DR4
DR5
DR4+5
C8
60
***
5
***
0
% sub-G0/G1
% sub-G0/G1
DR4
H460
20
siRNA (10nM)
SC siRNA
50
40
***
30
20
TRAIL
40
DR4+5 siRNA
30
20
***
10
10
0
No Drug
% sub-G0/G1
Receptor-specific
fluorescence
SC DR4 DR5
40
***
0
SC
SC
DR4
DR5 DR4+5
FT+TRAIL
-
+
-
SC
+
5µM cis
FT
Supplementary Figure 2. Role of death receptors in regulating TRAIL and FLIP siRNA induced cell death in NSCLC.
A Cell surface expression of DR4, DR5 and Fas in H460 cells transfected with 10nM control (SC), DR4, DR5 or Fas targeted siRNAs for 24 hours. Expression was
compared to an isotype control antibody. B Western blot analysis showing the expression of DR4 and DR5 in H460 cells transfected with 10nM SC, DR4 or DR5 targeting
siRNA for 24 hours. Equal loading was confirmed by analysing β-Tubulin expression. C Flow cytometry analysis of apoptosis in H460 cells treated with rTRIAL and cotreated with a TRAIL neutralising antibody (anti-TRAIL) for 24 hours. Apoptosis was assessed by analysing the sub-G0/G1 fraction of cells. The TRIAL neutralising
antibody was found to significantly reduce TRAIL-induced apoptosis in H460 cells. D Flow cytometry analysis of apoptosis in H460 cells transfected with 10nM SC, DR4,
DR5 and Caspase-8 (C8) siRNA for 24 hours followed by treatment with 5ng/mL TRAIL for 24 hours. Apoptosis was assessed by analysing the sub-G0/G1 fraction of
cells. DR4+5 and C8 siRNAs were found to significantly rescue TRAIL-induced apoptosis (*** denotes p,0.001, Student’s t-test). E Flow cytometry analysis of apoptosis in
H460 cells transfected with 10nM DR4 and/or DR5 siRNA for 24 hours followed by transfection with 1nM SC or c-FLIP targeted (FT) siRNA for 24 hours. Cells were cotreated with 2.5ng/mL TRAIL for the final 24 hours. F Flow cytometry analysis of apoptosis in H460 cells transfected with 10nM DR4 and DR5 siRNA for 24 hours followed
by transfection with 1nM SC or c-FLIP targeted (FT) siRNA for 24 hours. Cells were co-treated with 5µM cisplatin for 48 hours. Apoptosis was assessed by analysing the
sub-G0/G1 fraction of cells. DR4+5 siRNA was found to significantly inhibit apoptosis induced by TRAIL, FT siRNA + TRAIL, FT siRNA and FT siRNA + cisplatin (***
denotes p,0.001, Student’s t-test).
a
40
FT
35
25
20
15
10
5
30
FT
25
20
15
10
Con
2.5nM Taxol
5
40
SC
***
% sub-G0/G1
FT
25
20
15
10
5
0
Con
5nM Taxol
0nM
10nM Taxol
22.5
20.0
17.5
15.0
12.5
10.0
7.5
5.0
2.5
0.0
SC
5nM
10nM Taxol
BEAS-2B
H727
H157
% sub-G0/G1
10
0
0
Con
30
SC
FT
5
0
35
15
***
SC
% sub-G0/G1
***
SC
34LU
20
***
FT
% sub-G0/G1
% sub-G0/G1
30
% sub-G0/G1
35
b
A549
H460
SC
FT
15
10
5
0
Con
5nM Taxol
0nM
5nM
10nM Taxol
Supplementary Figure 3. Effect of FLIP silencing on taxol-induced apoptosis in NSCLC and normal lung cell lines.
A Flow cytometry analysis of apoptosis in NSCLC cells transfected with 1nM non-silencing control (SC) or FLIP-targeted (FT) siRNAs for 48h. Transfected cells were cotreated with taxol as indicated. The combined effects of FT siRNA and taxol were shown to be supra-additive (*** denotes p<0.001 as determined by two-way ANOVA).
B Flow cytometry analysis of apoptosis in BEAS-2B and 34LU cells transfected with 1nM SC or FT siRNA for 48h. Transfected cells were co-treated with taxol as
indicated. Apoptosis was assessed by analysing the sub-G0/G1 fraction of cells. The normal lung cell line models were not sensitized to taxol following FLIP silencing.