Alexis Alicea

Pinnacle Experiment
Dr. Stotz-Potter
Dexamethasone induced C2C12 cells caused no Significant difference
in DNA concentration, protein concentration, and cell diameter
Introduction
Dexamethasone is a synthetic glucocorticoid steroid that, when
administered over a long period of time, is known to cause muscle
atrophy in animal cells. Glucocorticoids are hormones that are
produced in the adrenal cortex released in the body to stop
inflammation as well as regulate other bodily functions. Synthetic
glucocorticoids are used to replace hormones, such as cortisol, if there
is an adrenal insufficiency and act as transcription factors for gene
expressions that are involved in production of proteins in the cell.
When dexamethasone is introduced to the cell, it formed a dimer
molecule inside the cell, moves into the nucleus via a nuclear pore
where it then acts as a transcription factor and attached to a code
promoter, and promotes protein degradation in cells. According to a
study done by Bonaldo P, Sandri M, 2013 dexamethasone cause the
genes that are expressed to code for ubiquitin in cells. During the
current experiment, there was no significant difference in protein
concentrations between the experimental group (Dexamethasoneinduced C2C12 cells) and the control group. Muscle atrophy was the
caused by four factors, increase in ubiquitin production in comparison
to muscle protein, increase in proteasomal ATP- dependent activity,
increase in protein breakdown that can be efficiently inhibited by
proteasome inhibitors, and increase in coding for ubiquitin (Bonaldo P,
Sandri M, 2013).
Results
Samples of one hundred different C2C12 cells for the control
group and the dexamethasone-induced cells were counted; the
diameter was measured between the nucleuses. There was shown to
have no significant difference in cell diameters. One tailed T-test when
assuming unequal variance is said to be 0.405 (P>0.05). Standard
error bars were placed using the standard error of the mean (Figure 1).
The average cell diameter for the control group was 5.15m and the
dexamethasone-induced cells were 5.13m. Using the standard error
of the mean error bars were determined for the control group to be
0.244 and the dexamethasone-induced cells were 0.229 (Figure 1).

5.16
5.15
5.15
5.14
Average cell diameter (m)

5.14
5.13
5.13
5.12
5.12
Control

Dexamethasone

Treatments

Figure 1 Means and standard error bars for cell diameters of both
control and dexamethasone induced C2C12 cells. There was no
significant difference (P>0.05)
Proteins were extracted, using mammalian protein extraction
reagent (M-PER) from both the control and dexamethasone-induced
C2C12 cell cultures. The concentrations of proteins for twelve trials
were measured; the averages of the protein concentrations for the
control group were 1.76 and for the dexamethasone-induced cells was
1.43 (Table 1).
Table 1 Average protein concentrations and standard deviations for all
twelve trials. There was no significant difference when using the
statistical T-test 0.35 (P>0.05) in protein concentrations between the
control and experimental groups.
Average Protein
concentration
Standard deviation of
protein

Control
1.76

Dexamethasone
1.43

2.23

1.73

There was no significant difference in protein concentration

between the experimental group and the control group (P>0.05). The
correlation between absorbance and bovine serum albumin (BSA) was
0.91 indicating a strong relationship (Figure 2). The individual protein
concentrations when using the linear regression equation for control
was 0.694 mg/ml and for the experimental 0.509.

1.4
f(x) = 0.79x + 0.19
R = 0.91

1.2
1
0.8
ABS (595 nm) 0.6
0.4
0.2
0
0

0.2

0.4

0.6

0.8

1.2

1.4

1.6

BSA Concentration (mg/mL)

Figure 2 Standard Curve for protein concentrations and absorbance at

595nm after deducting the blank from all absorbances. This graph was
based on the individual experiment. Protein concentrations were
determined using the linear regression equation.
Based on the results of the DNA concentration there was no
significant difference between the control group and experimental
group for all twelve trail (P>0.05); the p-value was 0.47. For the
individual DNA concentrations, it was determined to be 4g/ml for the
control group and 3g/ml for the experimental group. The average DNA
concentrations for all trials were 17.01g/ml for the control and 17.2g
for the experimental group. The purity of should lie between 1.8,
however in the current experiment it was determined for the control
group that was slightly above 1.9 and the experiment group fell within
standard range (Table 2).
Table 2 Purity of DNA using absorbance ratios for all twelve trials. There
was no significant difference (P>0.05) between the control and
experimental groups.
Absorbance (nm)
A260/A280
A260/A230

Control

Dexamethasone

2.0
0.31

1.5
0.38

Discussion
The purpose of this experiment was to determine the effects of
dexamethasone on C2C12 cells DNA concentration, protein
concentration, and cell diameter. The results show that there was no
significant difference in DNA concentration, protein concentration, and
cell diameter. It was expected that inducing differentiated C2C12 cells

with dexamethasone would cause down-regulation of DNA

concentration, which in turn would cause inhibition of protein, therefore
a lower concentration of the protein. It was also expected that the cell
diameter between nuclei would have significantly decreased. The
embryonic C2C12 cells were incubated, differentiated and, the
experimental group was then induced with dexamethasone.
Cell diameter was not significantly different between the
experimental and the control group. According to a study done by Quin
et al 2006 it was determined dexamethasone induced mice showed
reduced body weight, length and maximum diameter of the tibialis
anterior in the dexamethasone-induced cells by 21.68%, 13.05%, and
15.09% when compared to the control group (Qin et al, 2013). This
contradicts the current study, based on relative percent difference in
the two averages of cell diameter there was only a relative percent
difference of 0.39% (Figure 1). This indicated that there was no major
difference in the size in diameter between the control group and the
experimental group. When cells are exposed to dexamethasone for a
longer period and a higher dosage of dexamethasone, the cells
diameter may have more noticeable effects (Qin et al 2013). The
concentration of the current study was 0.10M.
Based on the results of the current experiment it was shown that
the C2C12 cells of both the control group and the experiment group
lacked purity (Table 2). At absorbance ratio 260/280, it can be
determined that there may have been protein contaminations.
Dexamethasone fell shortly below the standard value of 1.8 (Table 2).
Absorbance ratio of 260/230 determines the presence of phenolate ion,
thiocyanates and other organic compounds; the standard value for
uncontaminated DNA is 2.0-2.2. For the control group and
experimental group, the ratio at absorbance at 230, the value were
respectively 0.31 and 0.38 it can be determined that there were a
contaminants in both the sample. There was no indication of
particulate contaminants in the solution (Table 2). This may be affected
by the low concentration of the samples used, dexamethasone
concentration was 3.0g/ml and for controls concentration it was
4.0g/ml. The cell plates used to grow C2C12 cells, when checked on
the tenth day; the medium was a yellow color. The yellow color was
due to the medium being used up by the cells and in turn started to
become acidic some cells died and were floating in the medium while
others were alive. In comparison to class data, the value for DNA
concentration of the individual experiment for the control group and
the experimental group was lower than the average of the twelve trials
(Dex=17.2g/ml, con=17.1g/ml). Upon removing the supernatant, the
pellot of DNA was accidentally removed. DNA concentration was
expected to be reduced in the dexamethasone-induced cells. Based on
a study done by Ing et al, 2014 it was determined that gene expression
were suppressed in the stallions that received the dexamethasone shot

than those who did not. The experimenters injected 0.10 mg/kg into
four stallions of the same stature; the control group, with similar
stature, was injected with the same volume of saline. The decrease in
DNA concentration range from 75%-50% when introduced to
dexamethasone (Ing et al, 2014). High variations of DNA
concentrations may be due to not counting the cell concentrations in
each plate prior to extracting the DNA. There was no significant
difference in the concentration of DNA in the control when compared to
the experimental group; the p-value was 0.47 (p>0.05).
It was expected that the protein concentrations in
dexamethasone would be degraded due to that fact that
dexamethasone will attach to the glucocorticoid receptor in the
nucleus and act as a transcription factor for the degradation of proteins
by altering the expression of genes. During the experiment, it was
shown that there was no significant difference, p-value was 0.35
(p>0.05). The cell concentration was not measure prior to removing
extracting the proteins. When comparing the protein concentration of
the individual data in the current study to the data of all trails in the
current study it shown a high percentage of difference for the
dexamethasone-induced cell groups (93.0%), as for the control groups
the relative percent difference was, 37.0%. There was high variation in
the twelve trials due to the possibility that the volumes of cells after
differentiation were different in each trial. A study done by Menconi et
al, 2008, with a purpose to determine the difference on muscle atrophy
in correlation to protein degradation with increasing concentrations of
dexamethasone. During Menconi et al, 2008 study it was found that
depending on the concentration of dexamethasone induced cells,
period of time cells were introduced to dexamethasone and stage of
differentiation stage of cells determined the extent of muscle atrophy.
It was concluded that dexamethasone did cause muscle atrophy in the
mouse cell line (Menconi et al, 2008).
During the current experiment it can be concluded that the
concentration of dexamethasone used (0.10M) did not cause a
significant difference of C2C12 cells between the control group and
experimental group in DNA concentrations, protein concentrations and
cell diameter. Upon further research of this topic the concentrations of
dexamethasone should be increased to determine a difference in
muscle atrophy.

Bibliography
Bonaldo P, Sandri M. 2013. Cellular and Molecular Mechanisms of
Atrophy. Disease Models and Mechanisms 6 (3): 25-39.
Ing NH, Forrest DW, Riggs KP, Loux S, Love CC, Brinsko SP, Varner DD,
Welsh TH. 2014. Dexamethasone Acutely down-regulates genes
involved in steroidgenesis in stallion testes. Journal of Steroid
Biochemistry and Molecular Biology 143 (4): 451-459.
Menconi M, Gonnella P, Lecker S, Hasselgren P. 2008. Dexamethasone
and Corticosterone induced similar, but not identical, muscle
wasting responses in cultures L6 and C2C12 myotubes. Journal of
Cellular Biochemistry 105: 353-364.
Qin J, Du R, Yang Y, Zang H, Li Q, Liu L, Guan H, Hou J, An X. 2013.
Dexamethasone-induced muscle atrophy was associated with
Upregulation of Myostatin promotor activity. Research in
Veterinary Science 94 (1): 84-89.