1.

Introduction

Protein expression is defined as a process in which proteins are synthesized, modified and
regulated in living organism (Brown, 1981). Protein synthesis is mainly regulated by ribosomes
which act as RNA-based enzymes, commonly known as ribozymes which promote peptide bond
formation. Ribosomes are composed of 2 subunits: the ‘large' subunit (60S in mammals) and the
‘small' subunit (40S in mammals) in the cytoplasm. Amino acids are delivered to the ribosome in
conjugates with the proper tRNA, which has an anticodon that is corresponding to the amino
acids codon. These amino-acyl-tRNAs are initially recruited to the ribosome's acceptor site,
which is also known as A site and other two more tRNA-binding sites on the ribosome are P-site,
which binds peptidyl-tRNA, and the E-site, which binds a free tRNA. The process of translation
involves the presence of many distinct proteins known as translation factors that are not present
on the ribosome (Thomas, 2019). The three stages of mRNA translation are commonly used.
During initiation, the ribosome and the tRNA for the first amino acid residue, methionine, are
placed at the first (or "start") codon (AUG) of the mRNA. In elongation, the polypeptide chain is
assembled step by step as directed by the ORF of the mRNA. Termination occurs when the
ribosome detects a ‘stop' codon, resulting in the release of the synthesized polypeptide and
ribosomal subunits (Scheper et al., 2007). Regulation of protein expression is just as important as
transcriptional control. Dysregulation of this mechanism is linked to a variety of diseases,
including cancer and various neurological diseases. Mutations in regulatory mRNA sequence
elements in 5’ and 3’ untranslated regions (UTRs), such as upstream open reading frames
(uORFs), internal ribosome entry segments (IRESs), and micro-RNA (miR) target sites, as well
as altered expression of trans-acting protein factors, mutation in these all factors may results in
several diseases (Le Quesne et al., 2010).

1.1 Protein expression in cancer

eIF4E (Eukaryotic Translation Initiation Factor 4E) was the first initiation factor to be linked to
cell transformation. eIF4E has been found to be overexpressed in a variety of tumors, including
those of the breast, lung, bladder, colon, and prostate (Graff et al., 2008). eIF4GI is
overexpressed in lung and inflammatory breast cancer, while eIF4A is overexpressed in
melanoma and primary hepatocellular carcinoma. Other components of the eIF4F complex are
also linked to malignant transformation (De Benedetti and Rhoads, 1990). In other hand, most

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tumor cells depend on the transcription factor c-Myc for their growth and proliferation. The
expression of MYC oncogene is most frequently associated with the tumor aggression that
means elevated expression of c-Myc can promote tumor growth in wide range of tissues. In
tumor cells, expression of c-Myc at high level results accumulation of transcription factors on
promoter regions which then requires the transcription elongation factor P-TEFb, and results
transcriptional amplification, producing increased levels of transcripts within the
cell which further increase the gene expression (Lee and Young, 2013).

1.2 Role of c-Fos expression in human body

c-Fos expression in the central nervous system was initially identified by following seizure
activity and noxious stimulation. The fast and transient expression of c-Fos is caused by neuronal
excitement (Herrera and Robertson, 1996). Immunohistochemical techniques can detect the
specific c-Fos protein within neurons for 20-90 minutes after neuronal activation, and it
disappears in 4-16 hours afterwards. The expression of c-Fos-like genes could be a specific
marker for neuronal activity at the single cell level (Bullitt, 1990). c-Fos is a proto-oncogene that
is expressed in neurons when the cell receives voltage-gated calcium (Morgan and Curran,
1988). The proto-oncogene c-Fos encodes the nuclear phosphoprotein Fos, which forms a
dimeric complex with another protein called Jun, which has DNA sequence binding sites for
transcription factor activator protein-1 (AP-1). Other later genes and proteins essential to
cognitive, motor, and learning processes are controlled by the JUN family and Fos-related
proteins, which generate several potential combinations among its members (Barros et al., 2015).
Kindling is a permanent change in the CNS by which a repeatedly administered but initially
subconvulsive stimuli, either chemical or electrical, results in a generalized seizure. Two of the
most salient features of kindling which deserve special attention are the irreversibility of this
phenomenon and the anatomical specificity (Herrera and Robertson, 1996).

1.3 ImageJ
ImageJ is a free Java image processing software based on the Macintosh's NIH Image (Collins,
2007). ImageJ is an image analysis program widely used in the research of biological sciences
and its beyond (Rueden et al., 2017). The image analysis methods in ImageJ are developed for
the assessment of quantitative microscopical data analysis (Hartig, 2013). In
immunohistochemistry, it is widely used for the image analysis and for the quantification of

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protein expression (Collins, 2007). The ImageJ program which process the images and gives the
data like density which allow to compare multiple images for the analysis of expression (Malhi
et al., 2014).

2. Material and Methods

2.1 Equipment
Computer Specifications: A 64-bit operating system that has Windows 7 or greater, Mac OS X
10.11 or greater or Linux

2.2 Software:
1. ImageJ Software (Tiago Ferreira, Wayne Rasband), version 1.44
2. Graphpad Prism 5.0
2.3 Methodology:
2.3.1 Processing of Image in ImageJ software
At the first step, given images saved in separate folders according to group name. Open ImageJ
software then click on File and Open; now select the image which is to be analyzed. Click on
Image in menu bar and select Type and 8-bit then OK. Next, click Process in menu bar and click
on subtract background and OK. In next step; go into Image menu and Click on Adjust then note
the minimum and maximum threshold value for control group which to be same for all images in
each group then select the B&W color option and close that tab. Next step; go into Analyze
option in menu bar and click on set measurements and click check boxes (Area, Mean gray
value, integrated density, Limit to threshold and Display label) and click OK. Next; select
rectangular box in tool bar and draw on full image then go into Analyze menu and click on
Measure; results displayed as area, mean and integrated density in a table. Repeat the same
procedure for all images and data which displayed in a table; save as excel file. Values of
integrated density to be used for the data analysis through Graphpad Prism software (Malhi et al.,
2014) (Crowe and Yue, 2019) (Ferreira and Rasband, 2012).

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2.3.2 Data Processing and Analysis using Graphpad Prism software
Open Graphpad Prism software and select Column option then select Bar graph followed by plot
Mean with SD and click on Create. Next step; enter all values of integrated density in A,B,C
column according to groups and label the columns as A: Saline control, B: KA seizures and C:
GYKI treatment. Next; go into Analysis in menu bar and click on Analyze then select One-way
ANOVA in column analysis and click OK then select Tukey: compare all pairs of column in Test
name option and click OK. Results obtained which show the significance between all groups
(Motulsky, 2007) (Brown, 2005).

2.4 Statistical Analysis

Data is presented as Mean ± standard deviation (SD). Data is analyzed using One-way ANOVA
test followed by Tukey’s multiple comparison tests. The significance of results between each
group is calculated as p-value (P<0.05, n=5).

3 Results
3.1. c-Fos expression in CA1
c-Fos expression in CA1 was estimated as integrated density i.e. Saline control group (mean ±
SD: 282069.2 ± 144511.3, n=5), KA Seizure group (mean ± SD: 897429 ± 95956.4, n=5) and
GYKI treatment group (mean ± SD: 476902.2 ± 58606.4, n=5). c-Fos expression in KA induced
seizure group displayed significant increase as compared to saline control group (Saline control
vs. KA seizure; p<0.001), treatment with GYKI significantly prevented the c-Fos expression
(GYKI Treatment vs. KA seizure; p<0.001) (Fig. 1 & 2).

Figure 1 Effects of GYKI 52466 treatment on KA-induced c-Fos expression in the CA1. A (Saline control) shown
normal c-Fos expression while B (KA seizure) shown increased c-Fos expression and treatment with GYKI shown
inhibition of c_Fos expression in CA1.

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Table 1 c-Fos expression in CA1
Label Area Mean IntDen RawIntDen MinThres MaxThres
1 saline control 1 CA1.jpg 1132 123.825 178751 178751 0 185
2 saline control 2 CA1.jpg 1140 124.016 201559 201559 0 185
3 saline control 3 CA1.jpg 633 145.859 159724 159724 0 185
4 saline control 4 CA1.jpg 888 154.303 387522 387522 0 185
5 saline control 5 CA1.jpg 711 151.92 482790 482790 0 185
6 KA seizures 1 CA1.jpg 1335 160.759 946999 946999 0 185
7 KA seizures 2 CA1.jpg 8929 155.171 897968 897968 0 185
8 KA seizures 3 CA1.jpg 6702 165.113 758981 758981 0 185
9 KA seizures 4 CA1.jpg 14929 155.805 1016776 1016776 0 185
10 KA seizures 5 CA1.jpg 1331 160.811 866421 866421 0 185
11 GYKI precond 1 CA1.jpg 1187 116.545 561740 561740 0 185
12 GYKI precond 2 CA1.jpg 1191 116.767 464170 464170 0 185
13 GYKI precond 3 CA1.jpg 1195 171.978 427778 427778 0 185
14 GYKI precond 4 CA1.jpg 1391 167.856 508435 508435 0 185

Figure 2 c-Fos expression in CA1, Values are presented as Mean ± SD. Data calculated by using One-way ANOVA
followed by Tukey’s multiple comparison test [p<0.001(*P<0.05, ** P <0.01, *** P <0.001, **** P <0.0001).

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3.2 c-Fos expression in Dentate gyrus
c-Fos expression in Dentate gyrus was estimated as integrated density i.e. Saline control group
(mean ± SD: 75822.2 ± 70117.9, n=5), KA Seizure group (mean ± SD: 3508134 ± 704794.2,
n=5) and GYKI treatment group (mean ± SD: 93558.6 ± 40798.1, n=5). c-Fos expression in KA
induced seizure group displayed significant increase as compared to saline control group (Saline
control vs. KA seizure; p<0.001), treatment with GYKI significantly prevented the c-Fos
expression (GYKI Treatment vs. KA seizure; p<0.05) (Fig 3 & 4).

Figure 3 Effects of GYKI 52466 treatment on KA-induced c-Fos expression in the dentate gyrus. A (Saline control)
shown normal c-Fos expression while B (KA seizure) shown increased c-Fos expression and treatment with GYKI
shown inhibition of c_Fos expression in dentate gyrus.

Table 2 c-Fos expression in dentate gyrus

Label Area Mean IntDen RawIntDen MinThres MaxThres
1 saline control 1 DG.jpg 1139 156.39 178128 178128 0 202
2 saline control 2 DG.jpg 173 155.202 26850 26850 0 202
3 saline control 5 DG.jpg 824 145.019 119496 119496 0 202
4 saline control 4 DG.jpg 214 173.047 37032 37032 0 202
5 saline control 3 DG.jpg 104 169.279 17605 17605 0 202
6 KA seizures 1 DG.jpg 17325 157.767 2733308 2733308 0 202
7 KA seizures 2 DG.jpg 16431 180.564 2966852 2966852 0 202
8 KA seizures 3 DG.jpg 25741 169.615 4366052 4366052 0 202
9 KA seizures 4 DG.jpg 20896 161.841 3381838 3381838 0 202
10 KA seizures 5 DG.jpg 25222 162.264 4092620 4092620 0 202
11 GYKI precond 1 DG.jpg 826 133.057 109905 109905 0 202
12 GYKI precond 2 DG.jpg 280 137.621 38534 38534 0 202
13 GYKI precond 3 DG.jpg 945 156.683 148065 148065 0 202
14 GYKI precond 4 DG.jpg 406 182.498 74094 74094 0 202

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Figure 4 c-Fos expression in dentate gyrus. Values are presented as Mean ± SD. Data calculated by using One-way
ANOVA followed by Tukey’s multiple comparison test [p<0.001(*P<0.05, ** P <0.01, *** P <0.001, **** P
<0.0001).

4 Discussion
In images from saline control group, there was very little c-Fos expression in CA1, while
increased c-Fos expression was seen in KA seizure group and treatment with GYKI results in
significant inhibition of c-Fos expression In the dentate gyrus of the hippocampus, seizure
control rats had a lot of c-Fos expression. GYKI administration before KA administration
significantly reduced seizure-induced c-Fos expression.

5 Conclusion
c-Fos expression in CA1 and dentate gyrus seen as increased level in Kainic Acid induced
seizure group as compared to saline control group which indicates Kainic acid induced seizure
rats shown high neuronal activity while treatment with GYKI decreased the c-Fos expression in
kainic acid induced seizure group. This study can be concluded as the treatment with GYKI may
indicate as anticonvulsant which to be more explored by further animal studies.

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