Proliferation and Differentiation of C2C12 Cells – An Analysis of Protein Location and
Dexamethasone Inhibition
Rationale
Cells are composed of a wide array of proteins, each with its own function. Some of these
proteins are only active in the presence of certain nutrients and can result in different functionality of
the cell. This is especially apparent in skeletal muscle cells, which change from proliferation to
differentiation in a changing medium, thus requiring different proteins to achieve different structures.
The goal of this experiment was to see how the expression of PCNA and myosin heavy chain are
impacted by a change from cell proliferation to cell differentiation in C2C12 cells. It was hypothesized
that PCNA, a translational protein, would be more active in the cell proliferation medium, and that
myosin heavy chain would be more active in the differentiation medium, as shown by Clarke et al.
(2007). The effect of dexamethasone, a corticosteroid, was also investigated in this experiment.
Previous literature suggested that dexamethasone would impact the location of glucocorticoid receptors
(Man et al., 2013), and would also impact the proliferation and possibly differentiation of cells (Desler
et al., 1996).
Results
Before the cells could be seeded and incubated, the cell viability and density had to be
calculated. To do this, a 10-fold diluted sample of cells were stained using Trypan Blue and counted in
a hemocytometer (Figure 1). Cells that excluded Trypan Blue appeared clear and were counted as
viable. Cells that retained Trypan Blue were deemed non-viable, as the cell membrane had to be
ruptured to retain the dye. The cell viability was calculated to be 82.35%, and the cell density of the
original sample was calculated to be 2450000 cells/mL. For full calculations see Appendix 1.
To achieve consistent results, the same number of cells needed to be seeded in each sample.
10.2μL of suspended cell solution was added to each well plate to achieve 25000 cells per sample (see
Appendix 3).
The morphology and confluence of cells were analyzed between three different time periods:
one, four, and 14 days of incubation, and in either control or dexamethasone treated environments
(Figure 2). Day one in both conditions showed similar morphology with myoblast shaped cells
containing cytoplasmic spindles surrounding the nucleus. Though the cells in the dexamethasone
medium were harder to resolve against the dye, they also had similar confluence with approximately
30% of the sample well filled. The day four cells also had a similar myoblast morphology. Both cell
types proliferated to some extent, but the cells incubated in control conditions were denser than those in
the dexamethasone conditions. Day 14 showed differentiation due to a change in medium, from 10%
fetal bovine serum to 2% adult horse serum. Differentiation occurred as expected in the control treated
cells, showing elongated myotubules containing multiple nuclei. The dexamethasone treated cells
showed some differentiation, but at a significantly smaller degree than the control sample. The
microtubules in the dexamethasone sample were more narrow and harder to resolve against the
background.
The location and expression of proteins was also investigated using immunocytochemistry.
PCNA was observed during both proliferation and differentiation and was found to only be present in
cell nuclei during proliferation (Figure 3). Myosin heavy chain (MHC) was also observed during both
time points and was found to be primarily active in the cytoplasm of the forming myotubules during
differentiation, with very mild activity in the cytoplasm during proliferation (Figure 4). Glucocorticoid
receptors were also tagged and observed during proliferation in both control and dexamethasone
conditions (Figure 5). These receptors were found primarily in the cytoplasm when the cells were
incubated in control conditions but were found exclusively in the nucleus when treated with
dexamethasone.
The day 7 PCNA experiment was repeated a second time and found inconclusive results. Figure
6 shows PCNA as active during differentiation in both the cytoplasm and the nucleus, though it was not
expected to be present in the cells at all during this time point, and if it was it was expected to just be in
the nucleus. This inconsistent result could be due to incorrectly labelling proteins, perhaps by adding
rabbit anti-MHC primary antibody instead of rabbit anti-PCNA primary antibody to the well. The
results could also have been the result of cell membrane rupture due to rough handling of the cells
during washing, but due to the localization of DAPI on the nucleus this is unlikely.
Discussion
The Trypan Blue exclusion experiment showed a moderately high cell viability, which was
expected for this sample. A similar experiment by Liao et al., (2006) found the cell viability of a
C2C12 sample to be 89.6%, using the same Trypan Blue exclusion technique as this experiment. This
shows that the viability and density were counted accurately, which allowed for all the samples to be
seeded with the same number of cells.
When analyzing the cell morphology in both control and dexamethasone conditions, it was
found that dexamethasone affects proliferation and differentiation. The confluency of the
dexamethasone plate was significantly less than that of the control plate, showing an inhibition in
replicating which would prevent rapid proliferation (Desler et al., 1996). Desler et al. (1996) measured
proliferation in C2C12 cells after 4 days of incubation in DMEM, 10% FBS, and varying
concentrations of dexamethasone. They found that the greater the concentration of dexamethasone, the
greater the inhibition of proliferation. They also measured the cells ability to uptake thymidine and
found it also decreased as the concentration of dexamethasone increased. They hypothesized that
exposure to dexamethasone inhibited a cells ability to synthesize DNA, which resulted in a decrease in
proliferation.
Differentiation was also limited by dexamethasone, though to a lesser extent than proliferation.
Dexamethasone increases the concentration of MuRF1, an enzyme that interacts with and inhibits
myosin heavy chain (Clarke et al., 2007). Myosin heavy chain is a vital component of myosin
filaments, responsible for twisting and untwisting to regulate muscle movement (Lodish et al., 2016). It
also provides some structural components of myotubules, meaning that a decrease in myosin heavy
chain would decrease the size and development of these myotubules (Lodish et al., 2016). The results
of this experiment found that myosin heavy chain is primarily active during cell differentiation. The
decrease in differentiation in dexamethasone conditions could be a result of myosin heavy chain being
inhibited and not able to help form the myotubule. The limited differentiation could also be partially
due to the inhibition of proliferation, as this sample would have had less cells to form the final
myotubule structure.
PCNA was also studied during this experiment. PCNA is a transcriptional protein that holds
transcription enzymes onto DNA, allowing for rapid and more accurate DNA replication (Lodish et al.,
2016). It was found that PCNA is only active during proliferation and only in the nucleus. This aligns
with expected results, as PCNA helps regulate protein transcription in the nucleus, and transcription is
only needed in mass amounts during the proliferation stage.
Glucocorticoid receptors are hormone receptors usually found in both the nucleus and the
cytoplasm, and are part of a hormone-receptor complex that can regulate certain genes within the cell
when activated (Guyre et al., 1998). They are found in all cell types and during most stages of a cell’s
life cycle (Guyre et al., 1998), including during proliferation and differentiation in C2C12 myoblasts. It
was found that in regular conditions, glucocorticoid receptors are found in abundance in the cytoplasm
and in small concentrations in the nucleus. This is expected for normal functioning and non-activated
glucocorticoid receptors. When activated by dexamethasone, the receptors appeared to localize to the
nucleus. This is due to a disruption in nucleocytoplasmic shuttling (Man et al., 2013). Previous
experiments using human skin cells showed a similar result, with dexamethasone being translocated to
the nucleus and excluded from the cytoplasm in the presence of dexamethasone (Man et al., 2013).
Myogenic index is the percentage of muscle cell nuclei that are present in the myotubules
(Capitanchik et al., 2018). It is determined by staining myosin to determine where the myotubules are,
and by staining the cell nuclei (Capitanchik et al., 2018). Nuclei from random samples are then
counted, and the average percentage that is located in a myotubule is calculated. The myogenic index
gives an indicator for how well cell differentiation occurred; i.e. what proportion of cells fully
differentiated into mature muscle cells. The myogenic index can be influenced by several factors, such
as the presence of steroids, the quality and contents of the differentiation medium (e.g. varying the
percentage of horse serum), and how long incubation occurred for (Capitanchik et al., 2018). An
increasing myogenic index means increasing quality in differentiation.
It was discussed how myosin heavy chain is upregulated during differentiation, however there
are many other proteins that are upregulated in this time frame. One example is the Myo-D protein,
which is responsible for implementing the transcription of MRFs, specific muscle proteins that assist in
the formation of fully developed myotubules (Xun et al., 2007). Previous literature found that in C2C12
cells, this protein is more active during differentiation (Xun et al., 2007). MRFs are found in almost all
cells, and in certain conditions can be activated by Myo-D to convert non-muscle cells into myoblasts
(Xun et al., 2007).
Blocking serum is used in immunocytochemistry experiments to block non-specific binding
sites and prevents the antibodies from binding to them, which would make determining the location of
a specific protein much more difficult (Lodish et al., 2016). If the only blocking serum available was
normal mouse serum, this experiment would not work on C2C12 cells, as they come from mice.
However, if investigating a different cell line, normal mouse serum could be used in combination with
secondary, fluorescently tagged antibodies that come from mice. The primary antibody could be
developed in a similar fashion to this experiment, injecting the protein of interest into a rabbit. The
secondary antibody would be produced by injecting the primary antibody into a mouse and allowing it
do produce the secondary antibodies.
It is possible to label two different proteins in the same cell using immunocytochemistry,
however the antibodies must come from different species to allow them to be differentiated from one
another. The primary antibodies must come from two different species, as they must be targeted by
different secondary antibodies, and the immune system of the animal producing the secondary antibody
may not notice a difference between two primary antibodies from the same species. The secondary
antibodies could come from the same species, as long as two individual members of that species were
used to make the secondary antibodies. Since they would be targeting different animal primary
antibodies they should not interfere with one another, however you would have to use two different
fluorophores to distinguish the colour difference between proteins.
References
Capitanchik, C., Dixon, C., Swanson, S., Florens, L., Kerr, A., & Schirmer, E. (2018). Analysis of
RNA-Seq datasets reveals enrichment of tissue-specific splice variants for nuclear envelope
proteins. Nucleus, 9(1), 410–430.
Clarke, B., Drujan, D., Willis, M., Murphy, L., Corpina, R., Burova, E., … Glass, D. (2007). The E3
Ligase MuRF1 Degrades Myosin Heavy Chain Protein in Dexamethasone-Treated Skeletal
Muscle. Cell Metabolism, 6(5), 376–385.
Desler, M., Jones, S., Smith, C., & Woods, T. (1996). Effects of dexamethasone and anabolic agents on
proliferation and protein synthesis and degradation in C2C12 myogenic cells. Journal of Animal
Science, 74(6), 1265–1273.
Guyre, P. M., Munck, A. (1998) Glucocorticoids. Encyclopedia of Immunology, 2, 996-1001
Xun, J., S., Kim, J., Oh, M., Oh, H., Sohn, Y., … Whang, K. (2007). Opposite roles of MRF4 and
MyoD in cell proliferation and myogenic differentiation. Biochemical and Biophysical Research
Communications, 364(3), 476–482.
Liao, J., Kang, J., Jeng, C., Chang, S., Kuo, M., Wang, S., … Pang, V. (2006). Cartap-induced
cytotoxicity in mouse C2C12 myoblast cell line and the roles of calcium ion and oxidative stress on the
toxic effects. Toxicology, 219(1-3).
Lodish, H., Berk, A., Kaiser, C. A., Krieger, M., et al. (2016). Molecular cell biology. New York: W.H.
Freeman and Company.
Man, X., Li, W., Chen, J., Zhou, J., Landeck, L., Zhang, K., … Zheng, M. (2013). Impaired nuclear
translocation of glucocorticoid receptors: novel findings from psoriatic epidermal
keratinocytes. Cellular and Molecular Life Sciences, 70(12), 2205–2220.
Appendix
1. Cell Viability =
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑡ℎ𝑎𝑡 𝑒𝑥𝑐𝑙𝑢𝑑𝑒𝑑 𝑇𝑟𝑦𝑝𝑎𝑛 𝐵𝑙𝑢𝑒
𝑇𝑜𝑡𝑎𝑙 # 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠
Cell Density of original sample =
=
𝑥100% =
98 𝑐𝑒𝑙𝑙𝑠
21+98 𝑐𝑒𝑙𝑙𝑠
# 𝑜𝑓 𝑐𝑒𝑙𝑙𝑠 𝑡ℎ𝑎𝑡 𝑒𝑥𝑐𝑙𝑢𝑑𝑒𝑑 𝑇𝑟𝑦𝑝𝑎𝑛 𝐵𝑙𝑢𝑒
𝑉𝑜𝑙𝑢𝑚𝑒 𝑜𝑓 𝑠𝑒𝑐𝑜𝑛𝑑𝑎𝑟𝑦 𝑠𝑞𝑢𝑎𝑟𝑒𝑠
𝑥 100% = 82.35%
𝑥 𝑑𝑖𝑙𝑢𝑡𝑖𝑜𝑛 𝑓𝑎𝑐𝑡𝑜𝑟
98 𝑐𝑒𝑙𝑙𝑠
𝑥10 = 2450000 𝑐𝑒𝑙𝑙𝑠/𝑚𝐿
4𝑥10−4 𝑚𝐿
2.
Figure 1: C2C12 Cells in a Hemocytometer Stained With Trypan Blue. Viewed under an upright
light microscope at a total magnification of 100x. Cells outlined with a white halo (red arrow) are cells
that excluded Trypan Blue, meaning they were viable, and were clear when viewed under the
microscope. Those with a blue halo (yellow arrow) are cells that retained Trypan Blue, meaning they
were not viable, and were blue in colour under the microscope.
3. Volume needed to seed 25000 cells =
25000 𝑐𝑒𝑙𝑙𝑠
2450000
𝑐𝑒𝑙𝑙𝑠
𝑚𝐿
𝑥
1000𝜇𝐿
1𝑚𝐿
= 10.2𝜇𝐿
4.
Figure 2: C2C12 Cells Stained Using Hematoxylin and Eosin. Cells were fixed using 10% neutral
buffered formalin, viewed at 100x magnification using an inverted light microscope. The nuclei are the
round structures that are stained a dark purple with hematoxylin, and the cytoplasm is the surrounding
structure that has been stained a pinkish-purple using eosin. A and B were taken after one day of
incubation in DMEM containing 10% fetal bovine serum (FBS), with B also containing
dexamethasone. C and D were taken after incubating for 4 days in DMEM containing 10% FBS, with
D also containing dexamethasone. E and F were taken after 14 days, after incubating 4 days in DMEM
containing 10% FBS and 10 days in DMEM containing 2% adult horse serum and DMSO. F was also
treated with dexamethasone at day 5.
5.
Figure 3: C2C12 Nuclei Fluorescently Tagged With DAPI (A and D) and PCNA Fluorescently
Tagged With Alexa Fluor 488 (B and E). Viewed under an inverted fluorescence microscope with a
total magnification of 200x. Cells were fixed using 10% neutral buffered formalin and fluorescently
tagged for PCNA detection using rabbit anti-PCNA primary antibody (1:100 dilution) and Alexa 488conjugated goat anti-rabbit secondary antibody (1:500 dilution). C is a merged image of A and B and
shows the location of PCNA during cell proliferation (day 3). F is a merged image of D and E and
shows the location of PCNA during cell differentiation (day 7). Images taken by Dr. Lepp of Brock
University.
6.
Figure 4: C2C12 Nuclei Fluorescently Tagged With DAPI (A and D) and Myosin Heavy Chain
(MHC) Fluorescently Tagged With Alexa Fluor 488 (B and E). Viewed under an inverted
fluorescence microscope with a total magnification of 400x. Cells were fixed using 10% neutral
buffered formalin and fluorescently tagged for MHC detection using rabbit anti-MHC primary antibody
(1:100 dilution) and Alexa 488-conjugated goat anti-rabbit secondary antibody (1:500 dilution). C is a
merged image of A and B and shows the location of MHC during cell proliferation (day 3). F is a
merged image of D and E and shows the location of MHC during cell differentiation (day 7). Images
taken by Dr. Lepp of Brock University.
7.
Figure 5: C2C12 Nuclei Fluorescently Tagged With DAPI (A and D) and Glucocorticoid
Receptors Fluorescently Tagged With Alexa Fluor 488 (B and E). Viewed under an inverted
fluorescence microscope with a total magnification of 400x. Cells were fixed using 10% neutral
buffered formalin and fluorescently tagged for glucocorticoid receptor detection using rabbit antiglucocorticoid receptor primary antibody (1:100 dilution) and Alexa 488-conjugated goat anti-rabbit
secondary antibody (1:500 dilution). C is a merged image of A and B and shows the location of
glucocorticoid receptors during cell proliferation (day 3) in the absence of dexamethasone. F is a
merged image of D and E and shows the location of glucocorticoid receptors during cell proliferation
(day 3) in dexamethasone. Images taken by Dr. Lepp of Brock University.
8.
Figure 6: PCNA (Green) and DAPI (Red) in C2C12 Cells During Differentiation. Viewed under
an inverted fluorescence microscope with a total magnification of 400x. Cells were fixed using 10%
neutral buffered formalin and fluorescently tagged for PCNA detection using rabbit anti-PCNA
primary antibody (1:100 dilution) and Alexa 488-conjugated goat anti-rabbit secondary antibody
(1:500 dilution).