SUPPLEMENTARY MATERIAL
Material and Methods
Western blotting
Cell lysates were prepared using protease and phosphatase inhibitors including DTT
(1mM), phenylmethylsufonyl fluoride (1mM), phenylarsine oxide (5M), NaF (25mM), Na3VO4
(2mM), leupeptin (100M), pepstatin (2M), N-tosyl-Lys-chloromethyl ketone (2.8M), Ntosyl-L-phenylalanine chloromethyl ketone (2.7M), 4-(2-aminoethyl) benzenesulfonyl fluoride
hydrochloride (4.17M), chymostatin (2M), aprotinin (2M), antipain (2M), N-ethylmaleimide (5mM), and MG132 (40M). Protein content in the cell extract was measured with
micro-BCA assay (Bio-Rad Laboratories; Hercules, CA). The uniformity of transfer in the blot
was confirmed by Ponceau staining. The binding of the primary antibody to the membrane was
detected by secondary antibodies conjugated with horseradish peroxidase and a
chemiluminescence system (Amersham Bioscience/GE Healthcare; Piscataway, NJ).
Real time PCR
The reaction synthesis for cDNA consisted of 500 ng of RNA, 2.5 µl of 10X RT buffer, 1 µl
of 100 mM dNTP mix, 2.5 µl of 10X random primer and 1.25 µl of RT enzyme) and used
for real-time PCR (the reaction cocktail consisted of 100 ng of template cDNA, 1 µl of 5 µM
forward and reverse primers – selected using primer express software v2.0 – and 25 µl of
SYBR green PCR mix). The survivin primers used were SURVIVINFOR
1
(5’CACCACTTCCAGGGTTTATTC C 3’) and SURVIVINREV (5’ TCT
CCTTTCCTAAGACATTGCTAA GG 3’). Actin transcript was used as an internal control
for all the samples tested using the primers hACTINFOR (5’ CTCACTGAGCGAGGCTAC
AGCT 3’) and hACTINREV (5’ TTGATGTCGCGCACGATTT 3’). The survivin primers
used amplify an amplicon of 75 bp whereas the actin primers amplify a 62 bp amplicon.
At the end of the PCR reaction, the CT (threshold cycle) and Rn (normalized
intensity of reporter dye) values were obtained. The values obtained for survivin were
normalized with actin reference values and the ΔCT values were thus obtained. Each
ΔCT value obtained for the ritonavir-treated samples was compared to that of the
DMSO treated samples values – which was considered as the calibration value – and the
relative mRNA levels of survivin calculated with the following formula: Relative
mRNA level = (2-(corrected Ct)).
Results
Ritonavir inhibits the growth of the KRAS wild type lung adenocarcinoma line H838 but does not
synergize with gemcitabine or cisplatin
Ritonavir, alone or in combination with gemcitabine or cisplatin, was tested in the KRAS wild
type lung adenocarcinoma line H838. Similarly to what was observed in the KRAS mutant lines
A549 and H522, ritonavir was able to inhibit the growth of the H838 line exhibiting an IC50 of
35 M (Fig. S1). On the other hand, the H838 line was resistant to gemcitabine, which reduced
the cell growth to a maximum of 40%. The ritonavir/gemcitabine combination failed to show a
consistent synergism. In fact, such a combination was synergistic only at high concentrations of
2
ritonavir (35-70 M) and gemcitabine (0.2-.04 M), exhibiting an average CI of 1.2  0.5 (Fig.
S1). Cisplatin was more efficient than ritonavir in inhibiting the growth of the H838 line and
their combined action was of antagonistic nature (CI > 1) (Fig. S1). Overall, these results suggest
that while ritonavir alone is efficient in inhibiting the growth of KRAS wild type lung
adenocarcinoma, its use in combination with gemcitabine or ciplatin is not as beneficial as for
the KRAS mutant cell lines.
Figure Legends
Figure S1. Ritonavir inhibits the growth of the KRAS wild type lung adenocarcinoma line
H838, but its activity is not enhanced by gemcitabine or cisplatin. Ritonavir inhibited the
growth of the H838 line exhibiting IC50 of 35 mM. Ritonavir did not consistently synergize with
gemcitabine, while it exhibited antagonism with cisplatin.
3
Tables
Table S1. siRNA sequences used for targeting survivin, c-Src and STAT3.
Name
Molecular
Weight
Sequence
siControl
(non-targeting)
IIIIIIIV-
AUGAACGUGAAUUGCUCAA
UAAGGCUAUGAAGAGAUAC
AUGUAUUGGCCUGUAUUAG
UAGCGACUAAACACAUCAA
13358
13358
13358
13358
siSurvivin
(targeting
Human
BIRC5)
IIIIIIIV-
CAAAGGAAACCAACAAUAA
GCAAAGGAAACCAACAAUA
CACCGCAUCUCUACAUUCA
CCACUGAGAACGAGCCAGA
13343
13358
13388
13418
SiSrc
(targeting
Human Src)
IIIIIIIVIIIIIIIV-
GAGAACCUGGUGUGCAAAG
CGUCCAAGCCGCAGACUCA
CCUCAGGCAUGGCGUACGU
CCAAGGGCCUCAACGUGAA
CCAACGACCUGCAGCAAUA
GGAGAAGCAUCGUGAGUGA
CCACUUUGGUGUUUCAUAA
UCAGGUUGCUGGUCAAAUU
13403
13433
13433
13418
13403
13403
13358
13373
SiSTAT3
(targeting
Human
STAT3)
4
Table S2. Quantitation of ritonavir-induced G0/G1 arrest in NSCLC lines. Cells were treated
for 48 h with ritonavir, and G0/G1 arrest was evaluated by PI staining. Profiling of PI incorporation
was performed by FACScan analysis using ModFit software. The table shows the results of one of
three consistent experiments – results are expressed as percentage of cells in each phase.
DMSO (%)
Ritonavir (20 M) (%)
Ritonavir (40 M) (%)
G0-G1
S
G2/M
G0-G1
S
G2/M
G0-G1
S
G2/M
A549
38
51
11
61
31
8
79
16
5
H522
38
44
18
64
31
5
76
18
6
5
Table S3. Quantitation of ritonavir-induced alteration of cyclins, CDKs, Rb, phospho Rb,
p53, and p27 expression. Cells were treated for 48 h with IC50 of ritonavir, and Western blots
were performed with their protein extracts. Triplicate measurements were done by densitometry
analysis of Western blotting. Measurements exhibiting P values < 0.05, <0.01, or <0.001 are
marked with one, two or three asterisks, respectively. In all cell lines, GAPDH levels were
unaffected by ritonavir.
A549
H522
Fold-change at ritonavir
Fold-change at ritonavir
IC50
p
IC50
p
CDK2
0.73
*
0.30
*
CDK4
0.25
*
0.53
**
CDK6
0.23
**
0.56
**
Cyclin D1
0.58
***
0.84
**
Cyclin A
0.18
**
0.38
**
p-Rb
0.16
**
0.08
*
Rb
0.50
**
0.55
**
P53
1.22
n.s.
0.28
*
P27
1.98
**
1.78
**
6
Table S4a. Quantitation of ritonavir-induced inhibition of c-Src, STAT3 and their
phosphorilated forms, and survivin in NSCLC lines. Cells were treated for 48 h with IC50 of
ritonavir, and Western blots were performed with their protein extracts. Triplicate measurements
were done by densitometry analysis of Western blotting. Measurements exhibiting P values <
0.05 or <0.01 are marked with one or two asterisks, respectively. In all lines, GAPDH levels
were unaffected by ritonavir.
A549
H522
Fold-
Fold-
change at ritonavir
change at ritonavir
IC50
Phospho-c-Src
0.40
Total-c-Src
1.0
Phospho-STAT3
0.63
Total-STAT3
1.04
p
*
IC50
p
0.10
**
0.60
*
*
0.62
**
n.s.
1.00
n.s.
0.71
*
n.s.
Survivin
0.55
**
Cleaved PARP
1.5
*
7
3.02
*
Table S4b. Quantitation of siRNA-mediated reduction of c-Src, STAT3 and survivin. Triplicate
measurements were done by densitometry analysis of Western blotting. Measurements exhibiting P
values < 0.05 or <0.01 are marked with one or two asterisks, respectively.
A549
Fold-change
s-Src siRNA
H522
p
Fold-change
p
c-Src
0.24
**
0.47
**
STAT3
0.49
*
0.59
*
Survivin
0.64
**
0.41
*
STAT3
0.31
**
0.17
**
Survivin
0.78
n.s.
0.75
**
Survivin
0.44
**
0.45
**
STAT3 siRNA
Survivin siRNA
8
Table S5. Quantitation of ritonavir-, gemcitabine-, and ritonavir/gemcitabine-induced
reduction of survivin. Cells were treated for 48 h with IC50 of ritonavir, gemcitabine, or a
combination of the two drugs and Western blots were performed with their protein extracts.
Triplicate measurements were done by densitometry analysis of Western blotting. Measurements
exhibiting P values < 0.05 or <0.01 are marked with one or two asterisks, respectively. R stands for
ritonavir, G for gemcitabine, and R+G for the combination ritonavir/gemcitabine.
Survivin
Fold-change
p
R
G
R+G
R
G
R+G
A549
0.47
0.53
0.37
**
**
**
H522
0.51
0.91
0.47
**
n.s.
*
9