2014

CGAS AND STING EXPRESSION AND ITS

INFLUENCE ON THE PHOSPHORYLATION
OF IRF3 PROTEINS WITHIN 293 CELL
LINES

Weill Cornell Medical College

2014

Jonathan Daniels, Packer Collegiate Institute

Eric Lam, Dr. Erik Falck- Pedersen, Weill Cornell Medical College

Jonathan Daniels
C GAS A ND S TING E XPRESSION

cGAS and STING Expression and its Influence on the

Phosphorylation of IRF3 Proteins Within 293 Cell Lines
Jonathan Daniels, Packer Collegiate Institute
Eric Lam, Dr. Erik Falck- Pedersen, Weill Cornell Medical College
A BSTRACT
The cGAS-STING DNA Sensing Pathway is a recently discovered immune response with the
potential as a therapeutic tool against inflammatory diseases such as systemic lupus
erythematosus. In this study we are trying understand this pathway better. We tested this pathway
by over expressing STING, cGAS and the myc tag genes involved to see if this would cause the
production of a larger amount of interferon proteins, which would mean a more effective immune
response. In my work, I first over expressed cGAMP, the product of cGAS in 293 cells as a way of
jumpstarting the pathway in hopes of creating an abundance of IRF3 proteins. What I discovered
was a possible degradation of STING, halting to the pathway and preventing any immune response.
In order to counteract this imbalance I then overexpressed both cGAS and STING genes in hopes of
creating an abundance of the resulting immune response proteins. I concluded from the
experiments presented in this paper that cGAS and STING must be over expressed at the same time
for efficient increase in immune response proteins.

enters the cell, the nucleotidyl transferase,
INTRODUCTION
cGAS,s zinc thumb, which is positively
charged, attracts the negatively charged
foreign DNA. The two structures bind
Infections from pathogens, such as viruses
producing the protein, cGAMP. (Xiao and
and bacteria occur on a daily basis. Most of
Fitzgerald, 2013). cGAMP then activates the
these dont make us sick because our immune
STING protein which phosphorylates
system protects us; some pathogens,
interferon proteins, such as IRF3. These
however, can leave lasting effects. Viruses for
proteins either return to the nucleus signaling
example inject their nucleic acids into the cell.
for the production of interferon proteins or
Without detection, ultimately the viral DNA
leave the cell, signaling to neighbor cells to
could travel into our nucleus and lay dormant
mount a counter attack against the infection.
in our genome until it begins to replicate,
(Sun et al., 2013)
killing our cells. In order for our cells to

counteract these attacks they require a
The cGAS- STING Pathway has gained much
pathway that is able to recognize an infection.
attention recently because of its potential as a
The recently discovered cGAS-STING pathway
therapeutic tool against inflammatory
is one of our most common ways of detecting
diseases caused by viral infections. In my
foreign DNA. (Falck-Pedersen et al., 2014)
work at the lab I discovered that when only

expressing cGAS by introducing cGAMP to the
Every human cell contains a set of
cell, STING was degraded, and the pathway
mechanisms which, when a pathogen enters
therefore did not work. (Daniels, 2014) In
the cells, allows it to recognize the infection
hopes of bypassing the possibility of
and fight against it. The DNA- STING pathway
degradation in STING from over expressing
is based on the function of specific proteins
cGAS I plan to simulate the reactions a cell
that stop the proliferation of infection in the
would have in the presence of viral DNA by
cell. (Falck-Pedersen et al., 2014) For
transfecting the 293 cells with both cGAS and
example, when foreign DNA from a virus

Weill Cornell Medical College

2014

Jonathan Daniels
C GAS A ND S TING E XPRESSION

STING to try to increase the production of

immune response proteins (IRF3 expression).
H YPOTHESIS
Contrary to my previous result showing that
the degradation of STING is caused by the
excessive amounts of cGAMP present, I
hypothesize that the expression of both cGAS
and STING will cause phosphorylation of IRF3
proteins. Because STING is expressed, STING
wont degrade as quickly in the presence of
the cGAMP produced by the introduction of
cGAS. This should show a significant increase
in the quantity of phosphorylated IRF3
proteins present.
M ETHODS A ND M ATERIALS
To over express both cGAS and STING in 293
cells we first plated out our cells while we
expanded plasmids that would express cGAS,
STING and GFP. The GFP (green fluorescent
protein) was used as a visual aid in
identifying which cells were transfected. The
concentration of the plasmids was then
calculated and diluted to give them all the
same concentration. The 293 cells were
treated with a combination of a version of
STING, cGAS and GFP and were incubated for
18 hours. Proteins from the cells were
isolated. Once again, they were calculated for
concentration and diluted as such in dye.
Through western blot analysis the presence
of each protein was determined

Cell Line: Type 293 Cell lines were plated in a
12 well plate, 3x10^5 cells per well in 1ml of
DMEM solution overnight.

Plasmid Expansion: To create ample DNA
for transfection, 65L of competent DH5
Alpha cells were transfected with 1ul DNA
plasmid samples; pcDNA-HA-CAT, STING,
TRIP cGAS, myc cGAS 1.1, myc cGAS 2.2, TRIP
cGAS D227A, TRIP cGAS E225A and mouse
cGAS respectively. Sat on ice for 10 minutes
and were then heat shocked in 42C water
bath for 50 seconds and were chilled on ice

Weill Cornell Medical College

2014

for 5 minutes. Incubated cells in 900ul of LB

at 37oC with moderate agitation for one hour
and then plated 150ul of each on LB-Amp
plates which incubated overnight at 37C.
One cell colony from each was expanded to
3ml to LB-Amp and then to 2ml of each was
expanded into 23ml of LB+ Amp flasks.

DNA Purification: A Qiagen Plasmid Plus
Midi Kit was performed using the Standard
Protocol: High- Copy Plasmid (22ml).
Following this, the samples were nano-
dropped. In table 1 the concentration for each
sample is shown:

Plasmid Concentrat DNA:
DNA:
ion (ug/ul) RNA
Protein
Ratio
Ratio
260:280 260:230
GFP
4.0
n/a
n/a
pcDNA- 2.2984
1.89
2.32
HA-CAT
(tag)
Flag
1.9258
1.89
2.30
STING 3
Flag
1.2922
1.88
2.34
STING 8
TRIP
2.1121
1.89
2.89
cGAS
myc
2.0637
1.89
2.38
cGAS 1.1
myc
2.5572
1.89
2.37
cGAS 2.2
TRIP
2.1242
1.89
2.36
cGAS
D227A
TRIP
2.0628
1.89
2.38
cGAS
E225A
Mouse
1.7862
1.89
2.34
cGAS
Table 1: DNA Purifications: Each plasmid expressed
the above gene. Above illustrates the concentrations of
each, showing the presence of DNA versus RNA versus
proteins.

Cell Treatment: Illustrated below are the

samples we treated the 293 cells with, 1g of
each individual plasmid diluted in 20l of
Optimum for a total of 3g in 60l of Optimum.

Jonathan Daniels
C GAS A ND S TING E XPRESSION

Samples
Mock (3x)
Mock w/ Lipofectamine (3x)
GFP+STING + pcDNA- HA- CAT
GFP+ STING+ TRIP cGAS
GFP+ myc cGAS 1.1+ pcDNA- HA- CAT
GFP+ TRIP cGAS + pcDNA- HA- CAT
GFP+ STING+ myc cGAS 1.1
GFP+ STING+ myc cGAS 2.2
TRIP cGAS D227A + STING + GFP
TRIP cGAS E225A + STING + GFP
GFP+ STING + mouse cGAS
GFP+ pcDNA- HA- CAT (2x)

on a MMN Lowry program at a 562
wavelength. The data from the spectrometer
was then exported to a preset formula. This
gave the amount of each sample that is to be
added to the wells after being diluted in 4x
Loading Buffer for a total volume of 20ul.

Sample
Amount
Added
Mock
5.5
Mock w/ Lipofectamine
6.6
GFP+STING + pcDNA- HA- 6.8
CAT
GFP+ STING+ TRIP cGAS
9.3
GFP+ myc cGAS 1.1+ pcDNA- 7.9
HA- CAT
GFP+ TRIP cGAS + pcDNA- 9.2
HA- CAT
GFP+ STING+ myc cGAS 1.1
9.1
GFP+ STING+ myc cGAS 2.2
8.9
TRIP cGAS D227A + STING + 10.3
GFP
TRIP cGAS E225A + STING + 8.2
GFP
GFP+ STING + mouse cGAS
8.5
GFP+ pcDNA- HA- CAT (2x)
7.8

Tube
1
2
3
4
5
6
7
8
9
10
11
12

Table 2: Cell Treatment: To prepare the plasmids for

cell treatment we diluted combinations of each plasmid
into a tube. Lipofectamine was cell food we used as a
mock. pcDNA and TRIP served as our cGAS genes and
the myc tags were cGAS genes with a tag that we hoped
would allow us to view cGAS.

50L of LF and 200ul of Optimum were

added to each tube and then sat for one hour.
Each tubes content was placed in a cell well
and rocked every 15 minutes for even
distribution of plasmids.

Protein Harvest: Proteins were harvested 18
hours after treatment using inhibitors to
prevent degradation and frozen at -80oC for
one hour.

Protein Assay: Harvested proteins were
centrifuged at 13,000rpm, 4C for 20 minutes
to separate proteins from pellet. The
supernatant was placed in chilled tubes and
prepared for standard BIO-RAD Dc Protein
Assay to calculate the amount of each tube
that should be added to the western for equal
proportions. 5L of each sample was placed
on a spectrometer plate along with 25ul of
reagent A and S (50:1). Using duplicate
dilution series as a standard curve the
samples were read for their optical density.
The samples were run in a spectrometer and
1
2
3
4
5
6
7

Weill Cornell Medical College

2014

Table 3: Protein Assays: Based on the BIO-RAD Dc

Protein Assay we diluted each protein in dye and added
the above amount of each solution to our gels each an
equal amount of proteins in each.

Each sample was then prepared in 4x Loading

Buffer, adding 5ul of sample and 15ul of
buffer to make a 20ul stock. The samples
were then polarized at 75oC for 10 minutes
before being added to the wells based on the
amount above.

Western Blot: Table 4 displays the order in
which the samples were added.

10

11

12

13

Jonathan Daniels
C GAS A ND S TING E XPRESSION

GFP+
STING+
mouse cGAS

GFP+TRIP cGAS
E225A +STING

GFP+TRIP cGAS
D227A+STING

GFP+STING+myc
cGAS 2.2

Weill Cornell Medical College

2014

GFP+STING+myc
cGAS 1.1

The gels were created using the standard Bio-

Rad Protean II Cells equipment, 10x Running
and 4x Loading Gel. Gels were submerged
in1x Tris Glycine Buffer ph 8.3 and run at
150V for one hour.

Wet Transfer: In a Bio-Rad Trans-Blot Cell,
the gels were compressed between a PVDF
transfer membrane and run in the Bio-Rad
Protean II box for one hour at 100V.

Blocking: Membranes were blocked for one
hour in 15ml of 5% TBS-T Milk with light
shaking.

Detection: pTBK1 (dilution 1:2500), pIRF3
(1:2500) and STING (1:3500) were diluted
into 5ml of 5%BSA/TBS as the primary
antibodies. 4L of secondary anti Rabbit HRP
coupled antibody in 10ml of 5% milk/TBS-T
as the secondary, sitting for one hour.

HRP Enzymatic Detection Reaction: 3mL of
HRP chemiluminescence substrate were
added to the membrane and sat for five
minutes before being placed in cassette. Films
were processed in the basement film
machine.

Stripping: Membrane sat for 40 minutes in
20 mL of tripping buffer at 50C and then was
washed three times with TBS-T.

Secondary Detection: pSTAT1 (1:2000),
Actin (1:3500) and pP65 (1:2500) were
diluted in 5% milk/TBS-T as the primary. 4ul
of secondary anti Rabbit HRP coupled
antibody in 10ml of 5% milk/TBS-T as the
secondary, sitting for one hour.

GFP+myc cGAS
1.1+ pcDNA- HA-
CAT

GFP+STING+TRI
P cGAS

GFP+STING+pcD
NA- HA- CAT

w/

GFP+TRIP cGAS+
pcDNA- HA- CAT

GFP+pcDNA-
HA- CAT (2x)

Mock
Optimum

Mock

Ladder

Table 4: Western Blot Wells: Placement of proteins

along a 15 well gel.

Tertiary Detection: Myc (1:3500) was

diluted into 5% milk/TBS-T as the primary.
4ul of secondary anti Rabbit HRP coupled
antibody in 10ml of 5% milk/TBS-T as the
secondary, sitting for one hour.

A NALYSIS
For the first round of antibodies, pTBK1,
pIRF3 and STING, we found evidence that
expression of both cGAS and STING causes an
increase in the phosphorylation of IRF3
proteins. Our blot shows that for cells treated
with STING and cGAS (see Figure 1, wells 7, 9
and 13), STING is present, resulting in
phosphorylation of IRF3 and TBK1.
Compared to our negative controls (see wells
2-5) in which either only STING, cGAS or
foreign DNA was introduced, no signs of
activation occurred.

Our second set of antibodies included pSTAT,
Actin and pP65. Figure 2, wells 7, 9 and 13 all
show presence of pSTAT1 and pP65,
confirming that there is further activation of
interferon proteins in the cGAS-STING
Pathway when both cGAS and cGAMP are
expressed.

Our final test involved the myc antibody to
test the efficiency of the myc tags we created.
Results show that in samples treated with
myc 2.2 showed no signs of its presence,
while those exposed to myc 1.1 showed
evidence of its presence leading us to believe
there may be something incorrect about the
characteristics of the myc 2.2 tags.
D ISCUSSION

Jonathan Daniels
C GAS A ND S TING E XPRESSION

Working in the lab, whether it was learning

how to create gels or the long hours of data
analysis have been a buildup to todays result.
I began my time in the lab working on the
replication of the cGAS construct in hopes of
using this copy to follow and record the
influence this construct has on immune
responses. Having obtained a copy of the
construct we created clones that have myc
tags attached to the end of the cGAS sequence
in hopes of being able to track the cGAS
construct. To check its efficiency we
transfected the cGAS myc tags into cells
treated with cGAMP in hopes of jumpstarting
the cGAS-STING Pathway. After running
western blots on the harvested proteins we
noticed that the myc tag appeared on bands
that indicate the same 55kDa length as the
cGAS construct. Hopeful that this proved the
success of the myc tag efficiency without
damaging the cGAS function we continued on
to the experiment of over expressing both
cGAS and STING, hoping to track the results
with myc tags.

The data shows that there is still much to be
understood of the cGAS-STING Pathway. We
now have a better comprehension of the
necessary equilibrium between both cGAS
and STING. Without both of these crucial
factors the pathway degrades from the over
abundance of cGAS activation. Future
questions include the reliability of the myc
2.2 as a tag for cGAS seeing as how it showed
no presence when tested. Another possible
topic to delve further into is the effects of
introducing the AD virus when we express
both cGAS and STING. Understanding this
may lead to further insight into the factors
necessary in expressing the pathway on a
larger scale.

Weill Cornell Medical College

2014

I would like to thank Eric Lam for the many
long hours he spent teaching me and to Dr.
Erik Falck-Pedersen for allowing me to intern
in his lab. Without them I probably still
wouldnt know how to run a simple gel.
Thank you.
R EFERENCES
1. Lam, E., & Falck-Pedersen, E. (2014).
Unabated Adenovirus Replication
following Activation of the
cGAS/STING-Dependent Antiviral
Response in Human Cells. Journal of
Virology. Retrieved December 17,
2014, from
http://www.ncbi.nlm.nih.gov/pubme
d/25297994
2. Lam, E., Stein, S., & Falck-Pedersen, E.
(n.d.). Adenovirus detection by the
cGAS/STING/TBK1 DNA sensing
cascade. Journal of Virology. Retrieved
May
2,
2014,
from
http://www.ncbi.nlm.nih.gov/pubme
d/24198409
3. T. Sam Xiao and Katherine A.
Fitzgerald (2013) The cGAS-STING
Pathway for DNA Sensing. Molecular
Cell Minireview
4. Lijun Sun, Jiaxi Wu, Fenghe Du, Xiang
Chen and Zhijian J. Chen (2013) Cyclic
GMP-AMP Synthase Is a Cytosolic
DNA Sensor That Activates the Type I
Interferon Pathway. Science
5. Daniels,
J.
(2014).
cGAS
Overexpression in Inactive cGAS
Bearing RPE Cells. Unpublished
manuscritpt, Weill Cornell Medical
Center

Jonathan Daniels
C GAS A ND S TING E XPRESSION

R ESULTS
Figure 1: Detection of TBK1, IRF3, STING proteins in
the wells.

Figure 2: Secondary detection of pSTAT1, Actin, pP65

proteins in the wells.

Weill Cornell Medical College
2014

Figure 3: Tertiary detection of the Myc tag proteins on

our modified cGAS, myc tag 1.1 and 2.2.