1. No category

Knockdown of p53 by Accell self-delivering siRNA causes inhibition
of p53-dependent DNA damage response in IMR-32 neuroblastoma
cell line and β-amyloid toxicity in rat cortical neurons
Žaklina Strezoska, Tamara Seredenina1, Devin Leake, Annaleen Vermeulen
Dharmacon, now part of GE Healthcare, 2650 Crescent Drive, Suite #100, Lafayette, CO 80026, USA
1
Siena Biotech S.p.A., Strada del Petriccio e Belriguardo 35, 53100 Siena, Italy
Dharmacon™ Accell™ siRNA enables efficient delivery in a wide range of
cell lines and primary cells. Accell siRNA reagents carry a novel chemical
modication pattern that facilitates the delivery of siRNA without a need for
transfection reagents.
NTC Accell
siRNA
Day 0
The ability to modulate gene expression in neuronal cell lines and primary
neurons using Accell siRNA opens new opportunities for functional genomic
siRNA screens in the eld of neuroscience.
p53 Accell
siRNA
Assay for:
Cell viability
p53 and p21 induction
Day 3
CTB assay
4 µM camptothecin
Normalized Alamar Blue (ut=1)
Control
(PPIB)
Cell exposure to damaging agents induces rapid increase of p53 protein levels
in the nucleus, leading to the induction of its transcription targets which control
cellular responses such as cell cycle arrest, repair and apoptosis.
P53 pool
DNA damaging agents/ UV / hypoxia / neurotoxins
Apoptosis
10
0
NTC
100
50
25
10
0
ADM in the transfection mix
NTC
100
50
25
10
0
Camp 4 µM
700
NTC
TP53 siRNA 2
TP53 siRNA 1
TP53 pool
GAP
PPIB
NTC
TP53 siRNA 2
p53
500
500
400
400
4 µM Camp
70
100
80
60
40
20
0
NTC
siRNA
#
NTC siRNA
p53 siRNA
60
50
40
30
20
10
0
p53
siRNA
**
5 µm
10 µm
15 µm
% β-amyloid 1-42, 48 hours
** p < 0.01; # p < 0.05, t-test
• More than 60% down-regulation of p53 mRNA at 48 hours posttransfection by Accell SMARTpool reagent.
• Silencing of p53 resulted in a signicant increase of neuronal survival
as compared to NTC siRNA as measured by MTT viability assay.
• The strongest protective effect was observed at 5 μM β-amyloid.
Conclusions
200
200
100
100
0
0
200
200
400
400
600
800
1000
1200
600
800
1000
1200
Nuclear Intensity
Nuclear Intensity
1400
1400
400
400
600
600
1400
1400
250
250
150
150
p53 intensity
p53 intensity
TP53 siRNA 2
TP53 siRNA 1
TP53 pool
GAPD
PPIB
NTC
TP53 siRNA 2
TP53 siRNA 1
0
MTT
300
300
200
200
20
B.
Camp 1 µM Camp 0.25 µM
60
40
Confocal microscopy (Zeiss LSM 510) Blue staining (Hoechst)
indicates nuclei. Scale bar 10 μm.
100
100
5050
00
200
200
800
800
1000
1200
1000
1200
Nuclear Intensity
• Silencing of p53 in IMR32 cells by Accell siRNA caused an increase in cell
survival upon camptothecin treatment.
• HCA with Cellomics Multiplexed p53 and p21 Detection Kit showed a
decrease in the p53 and p21 activation following camptothecin treatment
in IMR-32 cells transfected with Accell siRNA targeting p53.
• Delivery of Accell siRNA was optimized in rat cortical primary neurons.
• Silencing of p53 in primary rat cortical neurons resulted in a signicant
increase of neuronal survival upon β-amyloid treatment.
• Accell siRNA delivery technology permits functional target validation in
neuroblastoma cell lines as well as primary cortical neurons.
p53
siRNA2
Nuclear Intensity
p53
siRNA1
p53 pool
GAPDH
110
100
90
110
80
100
70
90
60
80
50
70
40
60
30
50
20
40
10
300
20
10
0%
A.
qPCR
D.
No treatment
0
25
qPCR
qPCR
• Confocal microscopy reveals virtually all neurons are
positive for Accell GAPD Red siRNA (A).
• Detailed analysis showed that siRNAs are localised in the
cytoplasm in neuronal cell bodies and in neurites (B).
0.00
4 µM Camp
80
TP53 pool
B.
PPIB
0.25
TP53 pool
0
TP53 siRNA 1
50
NTC
0.5
transfection mix
P21 Intensity
100
Mean Avg Intensity Ch3
Viability–ADM-2%FBS
PPIB
0.25
p21 intensity
150
No treatment
p53 induction
Viability–ADM
NTC
0.50
600
600
Knockdown–ADM
Knockdown–ADM-2%FBS
0
p53 knockdown provides neuroprotective
effect from β-amyloid peptide in primary
rat cortical neurons
0.75
200
1.25
UT
TP53 siRNA 2
1.00
C.
250
NTC
p21 induction
Efficient target mRNA knockdown
in IMR-32 cells by Accell siRNA
Normalized target expression/viability (NTC=1)
GAP pool
HCA analysis: Reduction in p53 and p21 following
camptothecin treatment when p53 is silenced
A.
0.75
TP53 siRNA 1
Apaf-1
-1
Casp-9
1
NTC pool
Accell SMARTpool and two individual siRNAs targeting p53 cause a signicant
rescue from camptothecin-induced cell death. This is observed in the phase
contrast cell images and the Promega™ Cell Titer Blue™ (CTB) cell viability assay
at 72 hours post-transfection.
GAP
14-3-3σ
-
TP53 pool
Cells were treated with different
Camptothecin doses for the last 24 hours
GAPD
GAD45α
1.25
ADM
µM
PPIB
p21
Bax
10
Neurons from E18 rats at 4 DIV (days in vitro):
Accell GAPDH Red siRNA (1 μM); 48 hours.
mM
MEAN AvgIntensity Ch2
Cell cycle arrest/genetic repair/cell survival
1.50
No drug
P
Mdm2
25
Efficient delivery of Accell siRNA in primary rat
cortical neurons
The cells were analyzed for cell survival by CTB assay and for induction of p53
pathway by High Content Analysis (HCA) using Thermo Scientific™ Cellomics™
Multiplexed p53 and p21 Detection Kit.
No drug
p53
MTT
MTT
% ADM in the transfection mix
Optimal conditions identied (50: 50 ADM: NBM)
that provide maximum target silencing with high
cell viability.
Knockdown of p53 increases the survival
of IMR-32 after camptothecin treatment
p53 is a mediator of numerous
cell-damaging agents
110
100
90
110
80
100
70
90
60
80
50
70
40
60
30
50
20
40
10
300
20
100
50
10
%0ADM in the
100
50
% ADM in the transfection mix
To demonstrate the utility of Accell siRNA reagents in neuronal cells, the effects
of the down-regulation of the tumor suppressor p53 was examined. This gene
plays a pivotal role in mediating DNA damage-induced apoptosis as well as
conferring a protective effect from β-amyloid peptide-induced neurotoxicity.
Here we describe how application of Accell siRNA enabled the development
of a high content screening assay in IMR-32 neuroblastoma cells and a whole
culture cell viability assay in primary rat cortical neurons.
Conditions tested:
• 100% Neurobasal medium (NBM)
• 50:50 NBM and Accell Delivery Media (ADM)
• 75:25 NBM and ADM
• 100% ADM
Camptothecin
Day 2
Primary neurons have special media
requirements (Neurobasal medium with B27
supplements) so Accell siRNA delivery conditions
were optimized to determine the best conditions
for cell viability and target gene silencing.
GAPDH
expression,
GAPDH
expression,
% of NTC
% of NTC
However, most neuroblastoma cell lines and practically all primary neuronal
cells suffer from low transfection efficiency due to the refractory nature of
the cells to lipid-based transfection reagents. As such, application of siRNA
for inducing RNA interference (RNAi), has limited utility in these cell types; thus
limiting their usefulness for development of functional assays for screening
and discovery of novel disease-relevant genes.
Purpose: Determine if knockdown of p53 by Accell siRNA will cause suppression
of the p53-dependent DNA damage response pathway in IMR-32 cells upon
camptothecin treatment.
IMR-32 Neuroblastoma cell line
Viability
(% of untreated control)
Neuroblastoma cell lines and primary neuronal cultures are commonly used as
cellular model systems for studying cancer and neuronal development as well
as being highly relevant models for the study of neurodegenerative diseases.
Neuronal
viability,
Neuronal
viability,
% of Neurobasal
control
% of Neurobasal
control
Experimental design
Expression levels normalised
to β-action (% of control)
Introduction
Testing media compatibility for Accell siRNA
delivery in primary neurons
• IMR32 cells at 20 000 cells/well on Collagen IV plates
• 1 μM Accell siRNAs control pools (NTC, PPIB or GAPD) and Accell SMARTpool
or individual siRNAs against p53 delivered in Accell Delivery Media (ADM)
with or without addition of 2% serum
• Knockdown and viability assessed at 72 hours post-transfection
• UT = untreated with siRNA; NTC = Non-targeting control pool
Day 0: IMR32 cells transfected with different Accell siRNAs
Day 2: -/+ 4 μM Camptothecin (Camp) for 20 hours
gelifesciences.com/dharmacon
Day 3: cells fixed and stained with Thermo Scientific™ Cellomics™ Multiplexed p53
and p21 Detection Kit.
A.Mean nuclear intensities in channel 2 (p21)
B.Mean nuclear intensities in channel 3 (p53)
C.Intensity in channel 1 (Hoechst stain) vs the intensity in channel 2 for p21 nuclear
stain (N=500 cell)
D.Intensity in channel 1 (Hoechst stain) vs the intensity in channel 3 for p53 nuclear
stain (N=500 cell)
TM