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Supplementary methods:
Cell lines and culture conditions:
MIA PaCa-2 and PANC-1 were cultured in DMEM, and AsPC-1 and BxPC-3 were cultured in
RPMI 1640 (Invitrogen, Carlsbad, CA) supplemented with 10 % fetal bovine serum (FBS), 50units/mL penicillin, and 50 μg/mL streptomycin (Life Technologies Inc., Frederick, MD). LT-2
cells were cultured in pancreatic cell culture medium containing pancreatic cell culture
supplements (Millipore Biosciences). hTERT-HPNE cells were cultured as recommended by
ATCC. Cells were incubated in a humidified 5 % CO2 atmosphere at 37 °C.
Antibodies and chemicals:
Antibodies specific for XIAP, PARP, Caspase 3, survivin, AKT, pAKT, LC3, ATG-5, RIG-I,
Bcl-2, Mcl-1 (Cell signaling, Boston, MA), Bax (BD Biosciences, San Jose, CA), NOXA,
PUMA (Imgenex, San Diego, CA), MDA-5 (in house) (18), HRP-conjugated secondary
antibodies (Dako, Carpinteria, CA), -Actin (Novus Biologicals, Littleton, CO), were used in
this study. Additional materials: Transcriptor First Strand cDNA Synthesis Kit, In Situ Cell
Death Detection Kit, Fluorescein (Roche Applied Science, Indianapolis, IN); MTT cell growth
assay kit (Millipore Corporation, Billerica, MA). pXIAP and pSurvivin were from Origene,
Rockville, MD. A constitutively active AKT (myr-AKT) plasmid 10841 was obtained from
Addgene (Cambridge, MA).
Plasmid transfection
Plasmid transfection experiments used FuGene HD transfection reagent using the manufacturer’s
protocol (Roche, Indianapolis, IN). Briefly, plasmid/siRNA was mixed with FuGene HD reagent
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(1:3 ratio) in 500 µL of serum free medium and left for 30 minutes to allow complex formation.
The complex was added to 100-mm plates, which had 2.5 mL of serum-free medium (2 µg
plasmid per mL medium). After 6 hours transfection, complete medium was added, and cells
were cultured further with indicated treatments.
LC3 assay
We used a previous protocol with minor changes (29). Briefly, cells were cultured on 8-well
chamber slides, mock-treated or treated with [pIC]PEI for 48 hours, fixed, permeabilized and
blocked with 1% BSA (w/v) in PBS for 1 hour at 4 °C. Cells were incubated overnight at 4 °C
with anti-LC3B antibody followed by FITC-conjugated secondary antibody for 1 hour, washed
and mounted with anti-fade mounting solution with DAPI (Invitrogen, San Diego, CA), and
analyzed by fluorescence microscopy.
Supplementary Figure Legend:
Supplemental Figure 1. [pIC]PEI treatment increases the Sub G1 population in pancreatic
cancer cells. Pancreatic cancer cells were either mock-treated or treated with the indicated doses
of [pIC]PEI complex for 36 or 48 hours and cells were collected and stained with propidium
iodide and FACS analysis was done to determine DNA content. Sub G1 population was
tabulated and data presented as percent cells in Sub G1 phase. Results are representative of three
independent experiments.
Supplemental Figure 2. Inhibition of autophagy by 3-MA or caspase-mediated-apoptosis by
z-VAD rescues pancreatic cancer cells from [pIC]PEI-induced cell death. Pancreatic cancer
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cells treated with indicated doses of [pIC]PEI for 24 hours and then treated with 3MA or PANCaspase inhibitor (z-VAD) for further 24 hours. Cells were collected and Western blotting was
done for XIAP-, LC3- and PARP-specific antibodies. -Actin served as loading control. Results
are representative of three independent experiments.
Supplemental Figure 3. [pIC]PEI does not change XIAP and survivin at the mRNA level. MIA
PaCa-2 cells were treated with 0.5 g/ml of [pIC]PEI for the indicated time. RNA was isolated,
and Real Time PCR analyzed XIAP and survivin mRNA. Results are representative of three
independent experiments.
Supplemental Figure 4. Inhibition of XIAP degradation does not rescue pancreatic cancer
cells from [pIC]PEI-induced apoptosis. Pancreatic cancer cells treated with the indicated doses
of [pIC]PEI for 24 hours and then treated with the MG132 proteasomal inhibitor for an additional
24 hours. (A) Cells were collected and Western blotting was done for XIAP, LC3, and PARP. Actin served as loading control. (B) Cells treated as in (A) were fixed and stained for TUNEL
according to the manufacture’s protocol. TUNEL positive cells were photographed and counted
from 5 random fields for each treatment group and represented graphically. * p<0.01 vs [pIC]
alone in all the cell lines. Results are representative of three independent experiments.
Supplemental Figure 5. Effect of expression of XIAP alone, survivin alone and the
combination on [pIC]PEI-induced apoptosis. (A) Pancreatic cancer cells (~5000 cells) were
grown in a 8-well chamber slide and transiently transfected with XIAP for 24 hours and then
treated with either [pIC], PEI or [pIC]PEI for an additional 48 hours. (B) Pancreatic cancer cells
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(~5000 cells) were grown on a 8-well chamber slide and transiently transfected with survivin for
24 hours and then treated with either [pIC], PEI or [pIC] PEI for an additional 48 hours. (C)
Pancreatic cancer cells (~5000 cells) were grown in a 8-well chamber slide and transiently
transfected with XIAP and survivin in combination for 24 hours and then treated with either pIC,
PEI or [pIC]PEI for an additional 48 hours. The cells were fixed and stained for TUNEL
according to the manufacture’s protocol. TUNEL positive cells were photographed and counted
from 5 random fields for each treatment group and represented graphically.
Results are
representative of three independent experiments. * p<0.01 vs [pIC] alone in all the cell lines ;
**p<0.01 vs [pIC]PEI (2.0 g/ml).
Supplemental Figure 6. Effect of over-expression of AKT on [pIC]PEI-induced apoptosis.
Pancreatic cancer cells (~5000 cells) were grown in a 8-well chamber slide and transiently
transfected with myrAKT for 24 hours and then treated with either [pIC], PEI or [pIC] PEI for an
additional 48 hours. The cells were then fixed and stained for TUNEL according to the
manufacture’s protocol. TUNEL positive cells were photographed and counted from 5 random
fields for each treatment group and represented graphically. Results are representative of three
independent experiments. * p<0.01 vs [pIC] alone in all the cell lines ; **p<0.01 vs [pIC]PEI (2.0
g/ml).
Supplemental Figure 7. [pIC]PEI treatment inhibits mTOR and PKC-
activation in
pancreatic cancer cells. (A, B, C, and D) Pancreatic cancer cells were treated with either [pIC],
PEI or indicated doses of [pIC]PEI for 48 hours and cell lysates were subjected to Western
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blotting to detect pmTOR, mTOR, PKC- and pRB. Results are representative of three
independent experiments.
Supplemental Figure 8. In vivo cytoplasmic delivery of [pIC] using invivo jetPEI induces
apoptosis in pancreatic tumors. Mice were sacrificed 3 days after the last dose of [pIC] PEI and
tumors were fixed in formalin and embedded in paraffin. (A) Formalin-fixed paraffin-embedded
tissue sections were stained for TUNEL and representative images of the indicated treatment
groups are shown. (B) IHC analysis of XIAP, survivin, cleaved Caspase-3 and CD31 in
pancreatic tumor xenografts from different groups of mice.
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