Fsi Research Paper

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Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

MCF7 Breast Cancer Cells versus HCT116 Colon Cancer Cells
Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274,

ZNF277) in MCF7 Breast Cancer Cells versus HCT116 Colon Cancer Cells
Durganili Balasubramaniyan
Frontiers of Science Institute

Table of Contents
List of Figures/Tables .2
Abstract3
Literature Review/Introduction4
Methods/Materials...9
Results14
Discussion..21
Acknowledgments..22
References..23

List of Figures/Tables
Figure 17
Figure 28
Figure 3..17
Figure 4..17
Figure 5..17
Figure 6..17
Figure 7..18
Figure 8..18
Figure 9..18
Figure 1018
Figure 1119
Figure 1219
Figure 1319
Figure 1419
Figure 1520
Figure 1620
Figure 1720
Figure 1820
Figure 1921
Figure 2021
Table 1...15

I.
Abstract
The goal of this research project is to figure out the location of the proteins ZNF274,
ZNF277, and ZBTB24 in MCF7 cells (breast cancer line) and HCT116 cells (colon cancer cells).
There has not been much research done on these transcription factors so being able to figure out
the location of the proteins will be an advancement in the research of the proteins. For this
project, various processes will be executed to figure out the location of the proteins. Gateway
cloning, cell culture, transfection, western blot, and immunofluorescence are the steps that are
taken to execute the goal. Western blot gave information on if there were proteins in the samples.
Immunofluorescence used three different stains (DAPI, 488, and Sy3) to stain the nucleus,
proteins, and cytoskeleton. In the end, the western blot did not show good results. The anti-flag
western blot did not show the bands in the columns. The beta-tubulin western blot worked
slightly. There were bands that were slightly visible, but not very clear. The immunofluorescence
showed good results for the proteins ZNF274 and ZBTB24. There were no proteins visible for
ZNF277. What was interesting is that in the cancer line MCF7, the proteins for ZNF274 were
outside the cell, but for HCT116 the proteins were inside the cell. This potentially means that the
transcription factor plays some role in the development of cancer. For future research, the
transcription factors ZBTB24 and ZNF274 can be studied further to see their function in the
development of cancer. For ZNF277, researchers can see why the proteins were not produced
and what went wrong in the expression of those proteins.

II.
Literature Review and Introduction
The central dogma of biology states that from DNA, RNA is made, and from RNA,
proteins are made. Regulation of transcription is done by transcription factors. They bind to
regions of DNA and give RNA polymerase a place to bind to for transcription to begin.
Transcription is the process in which RNA polymerase copies the target DNA strand into mRNA.
Since DNA cannot leave the nucleus, this process is important in getting the information held in
the DNA out to the ribosomes. The mRNA can leave the nucleus so it can send the necessary
information to the ribosomes. The mRNA then goes on to ribosomes and is translated into
proteins with the help of tRNAs. The tRNAs have recognition sites for specific amino acids that
allow them to get the right amino acid, which is the building block of proteins. The creation of
the amino acids in the proper order creates the protein that the cell needs. The absence of
transcription factors means that RNA polymerase cannot bind to the DNA and thus cannot copy
the DNA into mRNA. This means that the proteins that the body needs cannot be created since
the mRNA is not created. So transcription factors play a major role in the regulation of the
manufacturing of proteins.
Cells only need to transcribe certain parts of the DNA since only certain proteins are
needed for the cell to function. This means certain transcription factors are turned on in certain
cells, so knowing which cells transcription factors are turned on in will allow one to know what
the function of the transcription factor is (Lee, T., Young, R., 2013). Two transcription factors
that are being studied are ZNF274 and ZBTB24. Both of these are zinc finger transcription
factors. The DNA segments that these transcription factors regulate is unknown and the overall
function of the transcription factors is not well understood either. That is the goal of this research

project, in that: we are trying to figure out what exactly these transcription factors regulate and
which cells they are located. Proteins can be found anywhere in the cell, but we predict the
transcription factors to be in the nucleus because that is where they complete their job. The two
transcription factors are categorized into zinc finger proteins. This means that they have a zinc
finer binding domain, so they can recognize similar ions. Zinc finger transcription factors are
useful in that they are encoded by small segments of DNA. This will allow researchers for more
control of the gene in that they can control the expression of the gene.
A mutation in ZBTB24 is known to cause a disorder in the human genome. This disorder
is very rare, but is known as ICF (immunodeficiency-centromeric instability-facial anomalies).
There are mainly respiratory and gastrointestinal problems associated with this disease (Greef,
2011). Also, the disorder is associated with facial anomalies. This means that the affected person
has anomalies such as hypertelorism, epicanthic folds, low set ears, and macroglossia.
Hypertelorism is a disorder in which the eyes are farther apart than the normal distance.
Epicanthic folds are when the upper eye is folded and this covers the inner corner of the eye.
Macroglossia is used to term long tongues (Bernuth, 2014). As well as causing this disorder,
ZBTB24 is known to play a role in B and T cell differentiation. This means that the gene and the
transcription factor play some role in the immune system. That is why mutations in ZBTB24
may have a link to problems with the immune system and causing the ICF disorder (Greef,
2011). The location of ZBTB24 is known to be on chromosome 6 at position 21 (uniprot.com,
2015). However, the exact type of cells that the transcription factors are turned on in is relatively
unknown, and the location of the proteins produced from these transcription factors is also
relatively unknown.

Figure 1 Chart of ZBTB24 and the affected cancer cell

ZNF274 is another transcription factor that is being studied. The gene that codes for this
transcription factor is associated with the disorder Denys-Drash Syndrome. This disorder affects
the kidneys and genetalia. The syndrome leads to kidney failure during childhood. People with
the disorder also form tumors in either one or both kidneys. A mutation in this gene does not
cause the Denys-Drash Syndrome, but does have some affect on the disorder. (University of
Copenhagen) Additional information that is known about ZNF274 is that the transcription factor
co-localizes with KAP1 and SETDB1. To co-localize means that the substances are found at the
same site. In this case, KAP1 and SETDB1 are both proteins that share the same site with the
transcription factor ZNF274. (Frietze, 2010) The protein KAP1 has a lot of various functions, but
one specific function is transcriptional regulation. SETDB1 is an enzyme that is primarily
regarded with chromatin modifications. (Frietze, 2010) The gene that codes for the ZNF274

transcription factor is located on chromosome 19, but like the ZBTB24 transcription factor, the
exact cells that the transcription factor is turned on in unknown (uniprot.com, 2015)
Figure 2 Chart of ZNF274 and affected cancer

Proteins are a large part of what we are made up of. In order to make proteins, the DNA
serves as the template to make RNA which is then used to make proteins. Transcription factors
are important proteins that help regulate the transcription of genes. The human genome project
has determined all the chemical sequences in the human body as well as mapping all the genes.
Using the human genome, researchers are able to identify target cells and identify the specific
cells that contain specific transcription factors. Another useful asset that researchers use to
identify the effects of certain DNA segments is plasmids. They are circular DNA that can be
placed in bacteria, like Ecoli, and replicate independently using the bacterias transcription and
translation machinery. They are not part of the bacterial DNA, though they can phenotypically
change the bacteria. When the bacteria replicates, the information that is coded in the plasmid is
shown through the phenotype of the bacteria. This will provide the researcher with information
on the function of the target gene. This is beneficial because the plasmid allows researchers to

see the effect of the DNA on the phenotype of the bacteria, so it gives researchers an idea of what
the function of the gene may be. Plasmids can also be selected for in both bacteria and
mammalian cells which makes them useful in processes like transfection. For example, if a
plasmid that has amipicilin resistance is inserted into bacterial cells, they will be selected for
since they have the resistance gene. Similarly, in mammalian cells, the same selection occurs if
the cells have the plasmid for blastocydin. This change in phenotype shows that the cells have
taken up the plasmid and successfully expressed the information in the plasmid. In this project,
we will be forcing the cells to express a plasmid that contains a flag tag gene. This flag tag gene
will allow us to see the proteins that were created by expressing the information in the plasmid.
Cancer cells will also be used in this project. Cancer cells divide rapidly and we can force
them to express genetic information. In this project, we are going to use MCF7 cells and
HCT116 cells. MCF7 cells are a line of breast cancer and HCT116 cells are a line of colon
cancer. Breast cancer is a caner that affects about 12 percent of women in America
(breastcancer.org, 2015). The survival rate is dependent on what stage the patient is in. If the
patient is in stage 1, they will have a 100% chance survival, but if they are in stage 4 the survival
rate is 22% (American Cancer Society, 2015). Colon cancer is the second leading cause of cancer
related death in America. (American Cancer Society, 2014) People in stage 1 have a 80-95
percent chance of surviving, but those in stage 4 only have a 10 percent chance of surviving.
(medicinenet.com, 2001) Both these cancer affect many people and create life-threatening
problems. Looking at the function of transcription factors in these cancer lines will allow us to
see if there is any connection between the cancer cell line and the protein that it expresses.
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III.
Methods and Materials
In the lab, HCT116 cells were first grown. In order to grow these cells, it is important to
make sure that the dead cells are off the plate and the live cells have fresh media available to
them. In order to do this, take the plate that the cells are growing on and take off all the old
media. Before the media is taken off, all the necessary liquids (DPBS, trypsin, and the media) are
pre-heated to 37 degrees Celsius. Then the liquid called DPBS, which washes the cell plate, is
added and this discards the dead cells that are just floating around on the top. In order to get the
dead cells that are stuck to the bottom of the plate off, trypsin is added. This has to be applied to
the cells and then incubated for 3-5 minutes. It is important to be precise with time on this step
since the trypsin can kill all the cells and that is counter intuitive. Once the trypsin has been
added and the cells have been incubated, the new media can be added and the cells are good for a
few days and can be incubated. The media is added in a ratio of 1mL of media for every 1 cm3.
For example, if a cell plate is 20 cm3, then 20 mL of media needs to be added to it.
PCR was the first major step that was completed. This process amplified the area of the
DNA that had the information for ZBTB24 or ZNF274 into mass amounts. The PCR products
were used to run a gel electrophoresis. The purpose is to make sure that there was the correct
DNA was in the samples; for example, ZBTB24 should show up as a certain band size based on
its base pair numbers. The size of the bands can be determined using a ladder that is also loaded
with the DNA samples. The ladder contains bands that are of known size and is used as a
reference for the other bands. The gel is made up of agarose. This enzyme allows DNA to move
through the gel and separate into the individual bands. The way the DNA strand move through
the gel is by a charge. The apparatus is plugged into a machine that sends a charge of a certain
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voltage into the gel. Since DNA is negatively charged, it moves towards the positive side. This
means that the DNA strands will move down the gel and separate themselves depending on size.
First a gel was made out of agarose and water and put the gel into the apparatus. Once the gel
hardened, the comb (that made the wells) was discarded and the samples of DNA were pipetted
into the wells. The first well was loaded with the ladder and then our DNA samples were loaded
in the other wells. A charge of 100 V was sent through the gel for about 20 minutes. Once that
was done, the gel was visualized under UV light to see if there was DNA in the samples. Bands
in each column meant good results because that says there was DNA in the sample. An
individual PCR was done for the genes coding ZNF277, ZNF274, and ZBTB24.
Once the PCR reaction was completed, PCR clean-up was done. This step extracted the
DNA from the primers and leftover reagents. This PCR step was done as a beginning step to
gateway cloning. This cloning step allows researchers to place a specific gene into a plasmid
vector through homologous recombination steps. This placement of a gene into a plasmid vector
allows for control of over expression in other cells through the process of transfection, which
was the next step performed.
Transfection forces the plasmid (containing a certain gene) into the cancer cells and the
aim is to have the cells express or overexpress the plasmid gene and produce its proteins. The
goal is to get the cells to overexpress the protein so they the proteins that are produced by the
transcription factors can be studied. For this, lipofectamine and optimum were main materials,
plus the target DNA. A master mix of 64.05 microliters of lipofectamine and 915 microliters of
optimum was created. Then the plasmid DNA was made so that it was at a concentration of 3
g/L. In the 6 well dish, each column had the same contents. Column A had ZNF274, column B
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had TCF7L2 (positive control), and column C was the blank which was an empty vector with no
gene insertion. For each, a different dilution was done since the DNA had different
concentrations. For A, there was 7.52 microliters of the DNA and 292.48 microliters of the
optimem. For B, there was 13.58 microliters of the DNA and 206.42 microliters of optimem. For
C, there was 12 microliters of the DNA and 288 microliters of optimem. At this point, there were
four tubes: one master mix and three tubes with the diluted plasmid. Once this was done, a
150:150 ratio of the two tubes (master mix and appropriate plasmid) was made for each cell well
and now there are 6 new tubes. Then these tubes were incubated for 5 minutes and after, 250
microliters of the corresponding tube to each well. Wells 1 and 4 had the gene ZNF274, wells 2
and 5 had the gene TCF7L2, and wells 3 and 6 had the pTRED- nFLAG. Then they were
incubated in 37 degrees Celsius overnight. The next day, DOX was added to the top rows, which
allowed transfection to occur. This means that the cells from the top should only express the
protein. The transfection process was completed multiple times. First, MCF7 pTR cells were
transfected but these died due to transfection errors. Next, HCT116 cells were transfected
without the DOX, but these died because the CO2 incubator broke. Finally, transfection with
MCF7 cells and no DOX was done and this transfection worked.
For protein extraction, lysis buffer was first made. It had 1 mL of RPA buffer, 5
microliters of pi, and 5 microliters of DMSF. The media was then removed from the 6 well dish
and washed twice with DPBS. 300 microliters of trypsin was then added to each well and
incubated for 4 minutes. Once the incubation time was done, 1 mL of media was added to each
well and the solution was homogenized. The homogenized cells were added to centrifuge tubes
and centrifuged for 1 minute. Once the solution was centrifuged, a cell pellet formed on the side
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and the liquid was around it. This required us to take out the liquid without disturbing the pellet.
Once the liquid was taken out, 1 mL of DPBS was added to each tube and homogenized and
centrifuged for 1 minute again. Once again, the media was extracted without disturbing the
pellet. Next, an estimation for the pellet sizes was needed. Tubes 2, 3, 4, and 5 were estimated to
be 10 microliters, tube 1 estimated to be 15 microliters, and tube 6 estimated to be 12 microliters.
This size was multiplied by 5 and that much of lysis buffer was added to each pellet. The tubes
were iced for about 30 minutes. Then the tubes were centrifuged 5 minutes and the liquid was
removed and transferred to another tube. The liquid, in this case, held the proteins. Once this was
done, the tubes were placed in the freezer.
The next step is western blot. This step lets us visualize the proteins on the gel. First, a
transfer buffer and running buffer was made. Then 40 microliters of the protein and 20
microliters of special staining dye were placed into a single tube. Then each tube was heated on
the hot plate for 5 minutes. Once the proteins have been heated, a pre made gel was placed into
the apparatus for western blot and filled up with running buffer. In the first well, the ladder was
added and in the rest of the wells, the proteins were added. This runs on 200 V for 45 minutes.
Once the gel has finished running, it was transferred to another apparatus. In order to do this, a
sandwich needs to be made. The bottom is a black sponge, then 3 filter papers, the gel, a
membrane, 3 filter papers, and another black sponge. This sandwich is enclosed in a case and put
in transfer apparatus. This time, the apparatus needs to be filled with the transfer buffer to the
top. This charge runs for 120 minutes at 60 V. Once the charge finished, the proteins from the gel
have been transferred onto the membrane. After the proteins were transferred onto a membrane,
they were coated with antibodies so they could be visualized. The first night the proteins were
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encoded with anti-flag antibodies. These were visualized after an hour. Once this visualization
was done, the membrane was coated with milk and kept in the cold room over night. The milk
serves as a coating for the proteins on the membrane and makes sure that other proteins that are
not wanted stay off the membrane. The next day, these were washed with PBS and re-coated with
anti-beta-tubulin to see a consistency between proteins extracted from all of the cell walls. Once
this visualization was done, the proteins were kept in the cold room with milk on them and they
were visualized few days later to get better results for the anti-flag antibodies.
Immunofluorescence was the last step and this was done to visualize the cell under a
microscope. There are three parts to this process: fixation, permeabilization, and
blocking/incubation. Fixation says to use 4% paraformaldehyde in PBS pH 7.4 for 10 minutes in
room temperature. Then, for permeabilization, the samples have to be incubated for 10 minutes
with PBS and with .1-.25% triton X-100. Next, the cell were washed in PBS 3 times for 5
minutes. Then for the last part, the cells incubated with 1% BSA, 22.52 mg/mL glycine in PBST
(PBS + 1% Tween) for 30 minutes. Once the incubation finished, the cells incubated in diluted
antibody in 1% BSA in PBST in a humidified chamber for one hour. These also could have been
left overnight at 4 degrees Celsius. Then, the decanted the solution was poured and washed with
PBS 3 times, 5 minutes each time. Once the washing was done, the cells were incubated with the
secondary antibody in 1% BSA for 1 hour at room temperature in the dark. Finally, the secondary
antibody was decanted and the cells were washed 3 times with PBS for 5 minutes each. Once all
these steps were done, the cells were mounted onto slides and taken to a confocal microscope to
visualize. Table 1 shows what antibodies were added to what cell wells. The difference in
antibody allowed for controls, as well as the right proteins to be stained.
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Table 1 Antibodies that were applied to the cells
IV.
Results
In order to visualize the proteins in the cell, immunofluorescence was done. The results
show that in HCT116, some of the proteins were expressed, but not all of the proteins were
expressed well. Figure 3 shows only a blue DAPI stain. Figure 4 is a picture of ZNF274 and it
shows proteins outside the nucleus. Figure 5 has a lot of proteins in the nuclei of the cells. The
red stain is very faint and that is because the Sy3 stain did not work very well. Figure 6 shows
ZNF277a and there was no specificity in the staining of the proteins. The green that is being
shown in the picture is most likely due to autofluoresecence, which is when living tissue emits a
green light, and this could just be the living tissue fluorescing, not the proteins. Figure 9 shows
ZNF277b and similar to ZNF277a there is no specificity in the proteins. There is a general green
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that covers the cell, which is the green from the autofluoresecence of the cells. Figure 10 and 11
show coilin and this expressed the proteins very well. This was expected because previous
research has proved that the proteins are located in the nucleus.
MCF7 showed similar results, but some were different. Figure 12 showed the correct
results in that the DAPI stain only showed up. Figures 13 and 14 show the ZNF274 proteins.
Figure 13 shows the faint outlining of the beta tubulin. Figure 15 shows the proteins for ZBTB24
are located in the nucleus. Figure 16 shows a faint green which means that the green is from the
autofluoresecence since the green is nonspecific and very faint. This was for the ZNF277a
protein. Figure 17 shows the ZNF277b protein and there is not much green in the cells. Figure 18
and 19 show the positive control, and as expected the proteins were located in the nucleus.
Figures:
Figure 3 HCT116 negative control

(no protein or beta tubulin)
Figure 4 HCT116, protein ZNF274
17

Figure 5 HCT116, protein ZBTB24
Figure 6 HCT116, protein ZNF277a
18

Figure 7 HCT116, protein ZNF277athe red stain of the cytoskeleton
Figure 8 HCT116, protein ZNF277athe green stain of the proteins
19

Figure 9 HCT116, protein ZNF277b
Figure 10 HCT116, Coilin (positive

control)
Figure 13 MCF7, protein ZNF274

Figure 11 HCT116, Coilin (positive
control)
20


Figure 12 MCF7 negative control
(no protein, no beta tubulin)
Figure 15 MCF7, protein ZBTB24

Figure 17 MCF7, protein
ZNF277b
21

Figure 16 MCF7, protein ZNF277a
Figure 18 MCF7, Coilin (positive

control)
Figure 19 MCF7, Coilin (positive

control)
Figure 20 Western Blot results for

the beta tubulin
22

V.
Discussion
In the immunofluorescence, figures 1 and 10 were accurate since only the DAPI stain
was applied to the cells. Protein ZNF274 was different between the two cancer lines. In HCT116,
the proteins were located outside the nucleus as shown in figure 2, but in MCF7 the proteins
were located in the nucleus as seen in figures 11 and 12. This change in location may have an
effect on the development of cancer. Protein ZBTB24 was expressed pretty well in both cancer
lines. Protein ZNF277 did not express well in either cell line.
Protein extraction actually did work and the Western Blot verified that we did achieve a
protein suspension. As seen in the picture, all the protein bands (we stained for Beta Tubulin and
this should be the same length for all proteins) are the same length. This control was made to
verify protein presence, and intensity/concentration of proteins on the membrane. As seen in the
picture, beta tubulin does not have a strong or intense band; this could have resulted from a small
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concentration of protein that was put in the gel and the antibodies were not specific enough to
pick up such a small concentration.
When we stained for anti-flag, the bands did not show up well in the first run and at all in
the second. The anti-flag was visualized a day after the proteins were put in the membrane and
this western blot was not very clear. We waited a few days and visualized again and there was
nothing on the membrane. This can be due to several reasons: maybe the proteins diffused back
into the membrane, the membrane is not known to hold accurate results after a while, or the
proteins diffused into the liquid over time. Either way, the western blot did not give exceptional
data for the genes we studied.
Acknowledgments
Thank you to Dr. Seth Frietze for mentoring this research project and providing the proper
materials to undergo the research processes. Thank you to Alexis Corcoran and Zach for assisting
with the research protocols and also mentoring this project as a whole. Thank you Preston Le and
Esme Fahnestock for the assistance in lab work. Thank you to Nicole Wood for assisting in the
written work throughout the project. Thank you to the Edward Madigan Foundation for
sponsoring this research for me and thank you to the Morris Family Foundation for sponsoring
my room and board here at UNC. Thank you to Lori Ball for coordinating The Frontiers of
Science Institute and The University of Northern Colorado for providing the opportunity to
conduct this research project
VII. References
Bernuth, Horst Von, Ethiraj Ravindran, Hang Du, Sebastian Frhler, Karoline Strehl, Nadine
Krmer, Lina Issa-Jahns, Borko Amulic, Olaf Ninnemann, Mei-Sheng Xiao, Katharina Eirich,
24

Uwe Klsch, Kathrin Hauptmann, Rainer John, Detlev Schindler, Volker Wahn, Wei Chen, and
Angela M Kaindl. (2014) Combined immunodeficiency develops with age in Immunodeficiencycentromeric instability-facial anomalies syndrome 2 (ICF2)
Frietze, S., O'geen, H., Blahnik, K., Jin, V., & Farnham, P. (2010). ZNF274 Recruits the Histone
Methyltransferase SETDB1 to the 3 Ends of ZNF Genes. PLoS ONE.
http://www.breastcancer.org/symptoms/understand_bc/statistics (Retrieved July 18, 2015)
http://www.cancer.org/cancer/breastcancer/detailedguide/breast-cancer-survival-by-stage
(Retrieved July 18, 2015)
http://www.cancer.org/cancer/colonandrectumcancer/overviewguide/colorectal-cancer-overviewkey-statistics (Retrieved July 18, 2015)
http://www.genecards.org/cgi-bin/carddisp.pl?gene=ZNF274 (Retrieved June 26, 2015)
http://genevisible.com/cancers/HS/Gene%20Symbol/ZBTB24 (Retrieved June 21, 2015)
http://genevisible.com/cancers/HS/Gene%20Symbol/ZNF274 (Retrieved June 21, 2015)
http://www.medicinenet.com/script/main/art.asp?articlekey=18349 (Retireved July 18, 2015)
http://www.uniprot.org/uniprot/O43167#subcellular_location (Retrieved June 21, 2015)
http://www.uniprot.org/uniprot/Q96GC6 (Retrieved June 21, 2015)
Jessica C. de Greef, Jun Wang, Judit Balog,Johan T. den Dunnen, Rune R. Frants, Kirsten R.
Straasheijm,Caner Aytekin, Mirjam van der Burg, Laurence Duprez, Alina Ferster, Andrew R.
Gennery, Giorgio Gimelli, Ismail Reisli, Catharina Schuetz, Ansgar Schulz, Dominique F.C.M.
Smeets, Yves Sznajer, Cisca Wijmenga Marja C. van Eggermond, Monique M. van Ostaijenten Dam, Arjan C. Lankester,Maarten J.D. van Tol, Peter J. van den Elsen, Corry M. Weemaes,
and Silvre M. van der Maarel (2011) Mutations in ZBTB24 Are Associated with
Immunodeficiency, Centromeric Instability, and Facial Anomalies Syndrome Type 2
Lee, Inh Tong, Young, A. Richard (2013) Transcriptional Regulation and Its Misregulation in
Disease
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1

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274,

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Literature Review and Introduction

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 1 Chart of ZBTB24 and the affected cancer cell

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 2 Chart of ZNF274 and affected cancer

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Methods and Materials

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Table 1 Antibodies that were applied to the cells

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 3 HCT116 negative control

Figure 4 HCT116, protein ZNF274

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 5 HCT116, protein ZBTB24

Figure 6 HCT116, protein ZNF277a

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 7 HCT116, protein ZNF277athe red stain of the cytoskeleton

Figure 8 HCT116, protein ZNF277athe green stain of the proteins

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 9 HCT116, protein ZNF277b

Figure 10 HCT116, Coilin (positive

Figure 13 MCF7, protein ZNF274

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 12 MCF7, protein ZNF274

Figure 15 MCF7, protein ZBTB24

Figure 14 MCF7, protein ZNF274

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Figure 16 MCF7, protein ZNF277a

Figure 18 MCF7, Coilin (positive

Figure 19 MCF7, Coilin (positive

Figure 20 Western Blot results for

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

Analysis and Comparison of Unknown Transcription Factors (ZBTB24, ZNF274, ZNF277) in

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