Investigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Additionally, the expression of the apoptosis-regulating proteins Bax, Bcl2, and p53 are presented

Darin Bloemberg

Joe Quadrilatero

Data in Brief

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Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure

AbstractInvestigating cell death signaling using cell culture is commonly performed to examine the effects of novel pharmaceuticals or to further characterize discrete cellular signaling pathways. Here, we provide data regarding the cell death response to either cisplatin or A23187 in sub-confluent C2C12 cells, by utilizing several concentrations and incubation times for each chemical. These data include an assessment of the activation of the proteolytic enzymes caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Additionally, the expression of the apoptosis-regulating proteins Bax, Bcl2, and p53 are presented

Bloemberg, Darin

Quadrilatero, Joe

Elsevier - Publisher Connector 

Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure 

Contents lists available at ScienceDirect
Data in Brief
Data in Brief 7 (2016) 1024–1030S
M
T
H
http://d
2352-34
(http://c
n Corr
N2L3G1
E-mjournal homepage: www.elsevier.com/locate/dibData articleCaspase activity and apoptotic signaling
in proliferating C2C12 cells following cisplatin
or A23187 exposure
Darin Bloemberg, Joe Quadrilatero n
Department of Kinesiology, University of Waterloo, Waterloo, Ontario, Canadaa r t i c l e i n f o
Article history:
Received 16 December 2015
Received in revised form
1 March 2016
Accepted 5 March 2016
Available online 12 March 2016x.doi.org/10.1016/j.dib.2016.03.032
09/& 2016 The Authors. Published by Else
reativecommons.org/licenses/by/4.0/).
espondence to: Department of Kinesiolog
, Canada. Tel.: þ1 519 888 4567; fax: þ1 5
ail address: jquadril@uwaterloo.ca (J. Quada b s t r a c t
Investigating cell death signaling using cell culture is commonly
performed to examine the effects of novel pharmaceuticals or to
further characterize discrete cellular signaling pathways. Here, we
provide data regarding the cell death response to either cisplatin
or A23187 in sub-conﬂuent C2C12 cells, by utilizing several con-
centrations and incubation times for each chemical. These data
include an assessment of the activation of the proteolytic enzymes
caspase-3, caspase-8, caspase-9, calpain, and cathepsin B/L. Addi-
tionally, the expression of the apoptosis-regulating proteins Bax,
Bcl2, and p53 are presented.
& 2016 The Authors. Published by Elsevier Inc. This is an open
access article under the CC BY license
(http://creativecommons.org/licenses/by/4.0/).Speciﬁcations tableubject area Biology
ore speciﬁc
subject areaApoptosis, caspasesype of data Graphs and Figures
ow data was
acquiredSpectroﬂuorometry, immunoblotting, microscopyvier Inc. This is an open access article under the CC BY license
y, University of Waterloo, 200 University Ave. West, Waterloo, Ontario
19 885 0470.
rilatero).
D
E
E
D
D. Bloemberg, J. Quadrilatero / Data in Brief 7 (2016) 1024–1030 1025ata format Analyzed
xperimental
factorsC2C12 cells were treated with cisplatin or A23187xperimental
featuresCells were administered cisplatin or A23187 and collected at several time points.
They were then prepared for spectroﬂuorometric assessment of proteolytic
enzyme activity or were analyzed for apoptosis-related protein expression using
immunoblotting. Brightﬁeld microscope images were also acquired for qualitative
assessment of cell morphology.ata source
locationUniversity of Waterloo, Waterloo, Ontario, Canadaata accessibility All data are provided with this articleD
Value of the data
 The data describe the cell death response in proliferating C2C12 cells following exposure to several
concentrations and incubation periods with either cisplatin or A23187.
 Provides data regarding the speciﬁc pathways of cell death activation in C2C12 cells to either
cisplatin or A23187.
 The data demonstrate that cell death in C2C12 cells by cisplatin involves signiﬁcant activation of
p53 and caspases, while A23187 involves caspase-independent mechanisms.1. Data
Two key signals which regulate the induction of apoptosis are DNA damage and calcium (Ca2þ)
[1,2]. Despite the common use of cisplatin (CisPL) and Ca2þ ionophores such as A23187 to induce
apoptosis in cell culture experiments, limited evidence exists in C2C12 cells. Here, we present data
describing the cell death response in sub-conﬂuent C2C12 cells exposed to CisPL or A23187 (Fig. 1).
1.1. CisPL-induced apoptotic signaling in C2C12 cells
Beginning with the previously used concentrations [3,4], C2C12 cells were administered CisPL in
increasing doses and intermittently collected over a period of 24 h (Figs. 2 and 3). Caspase activity
was spectroﬂuorometrically measured using ﬂuorogenic substrates speciﬁc for each enzyme [5,6].
CisPL treatment caused time-dependent increases (po0.05) in the activity of caspase-3 and caspase-
9 (Fig. 2A and B). For caspase-3 and caspase-9, 25 mM and 50 mM CisPL induced larger (po0.05)
elevations in enzyme activity than 100 mM (Fig. 2A and B). However, despite increased (po0.05)
caspase-8 activity at 16 h and 24 h compared to 8 h, 50 mM and 100 mM CisPL doses reduced (po0.05)
caspase-8 enzyme activity (Fig. 2C). Data regarding the levels of apoptosis-regulating proteins at the
16 h time point also indicated concentration-dependent changes (Fig. 3). Here, CisPL elevated
(po0.05) the Bax/Bcl2 ratio, the amount of cleaved caspase-3, p53 protein levels, and the ratio of
cleaved/uncleaved PARP protein (Fig. 3A–C). Of note, 50 mM CisPL dramatically increased (po0.05)
p53 protein content above that caused by other concentrations. Despite observing the most sig-
niﬁcant changes to apoptotic markers with 25 mM and 50 mM CisPL, qualitative assessment of
brightﬁeld microscope images of Giemsa stained cells indicated that 100 mM had the greatestFig. 1. Overview of experimental treatment protocol.
Fig. 2. Caspase activity in response to CisPL treatment. (A) CisPL induced concentration- and time-dependent changes in
caspase-3 activity. (B) Similar effects were observed for caspase-9. (C) CisPL administration did not elevate the activity of
caspase-8. Values are expressed relative to CTRL (vehicle-treated) 8 h, which has been arbitrarily given a value of 1.0. *po0.05,
signiﬁcantly different than 8 h (main effect of time); $po0.05, signiﬁcantly different than 8 h and 16 h (main effect of time).
Main effects (po0.05) for CisPL dose are indicated with superscript letters, where groups with different letters are signiﬁcantly
different from each other.
D. Bloemberg, J. Quadrilatero / Data in Brief 7 (2016) 1024–10301026negative impact on cell conﬂuence and morphology (Fig. 3D), perhaps suggesting non-apoptotic
mechanisms of cell death at this dose.1.2. A23187-induced cell death signaling in C2C12 cells
Sustained high levels of cytosolic Ca2þ can activate apoptotic signaling mechanisms [7]. While
several ways of mimicking ER/Ca2þ-stress exist, ionophores allow speciﬁc alterations to ion levels
without affecting accessory cellular protein functions. A23187 is a partially-selective Ca2þ ionophore
widely used to increase cytosolic Ca2þ levels in cell culture. Previously, 1 mM A23187 treatment for 2 h
was shown to elevate calpain activity 3-fold in proliferative C2C12 cells, while increasing con-
centrations caused progressive drops in cell viability over 6 h [8]. Here, varying concentrations of
A23187 were administered to cells over 6 h in order to assess the appropriate conditions for causing
Ca2þ-induced apoptotic signaling in sub-conﬂuent C2C12 cells. These data demonstrate that A23187
treatment did not cause caspase-3, 8, or 9 activation at either time point (Fig. 4A–C). In fact,
10 mM and 15 mM doses generally reduced (po0.05) the activity of these three proteolytic enzymes
(Fig. 4A–C). While 5 mM A23187 slightly elevated (po0.05) calpain activation (Fig. 4D), two higher
concentrations reduced (po0.05) calpain enzyme activity (Fig. 4D). Assessing the lysosomal hydro-
lase cathepsin B/L indicated that activity was generally higher (po0.05) at 3 h compared to 6 h,
where 5 mM and 10 mM doses increased (po0.05) activity, while 15 mM reduced (po0.05) activity,
particularly at the 6 h time point (Fig. 4E). Finally, 5 mM A23187 appeared to moderately activate
upstream apoptotic signaling as indicated by an elevated (po0.05) Bax/Bcl2 ratio (Fig. 5A and D).
However, higher concentrations reduced (po0.05) the Bax/Bcl2 ratio, p53 protein (Fig. 5B and D), and
levels of pH2AX (Fig. 5C and D), a marker of DNA damage. Despite this relative lack of apoptotic
signaling activation, brightﬁeld microscope images of Giemsa stained cells demonstrated dramatic
Fig. 3. Changes to expression of apoptotic signaling proteins in response to CisPL at the 16 h time point. (A) All CisPL treat-
ments elevated the Bax/Bcl2 ratio, while 25 mM and 50 mM doses signiﬁcantly increased cleaved caspase-3 levels. (B) CisPL
elevated p53 protein content and the relative amount of cleaved PARP. (C) Representative immunoblots. (D) Brightﬁeld
microscope images of Giemsa stained CTRL (vehicle-treated) and CisPL treated cells. Values for CisPL-treated groups are
expressed relative to CTRL groups, which have been arbitrarily given a value of 1.0. *po0.05 compared to CTRL, $po0.05
compared to CTRL and 100 mM, #po0.05 compared to all other groups. Scale bar represents 100 mm.
D. Bloemberg, J. Quadrilatero / Data in Brief 7 (2016) 1024–1030 1027impacts on cell morphology caused by 10 mM and 15 mM A23187 compared to vehicle-treated CTRL
cells (Fig. 5E).2. Experimental design, materials and methods
2.1. Cell culture and experiment
C2C12 mouse skeletal muscle myoblasts (ATCC) were cultured as previously performed [6,9] on
polystyrene cell culture plates (BD Biosciences) with media consisting of low-glucose DMEM (Ther-
moFisher) with 10% fetal bovine serum (FBS; ThermoFisher) and 1% penicillin/streptomycin (Ther-
moFisher). Upon reaching 60–70% conﬂuence, cells were administered either cisplatin (CisPL; Enzo
Life Sciences) or the calcium ionophore A23187 (Sigma Aldrich), or maintained in a control (CTRL)
condition which was given an equal volume of vehicle (saline for CisPL experiments; DMSO for
Fig. 4. Proteolytic enzyme activity induced by A23187. A23187 had concentration-dependent but time-independent effects on
the activity of caspase-3 (A), caspase-9 (B), caspase-8 (C), and calpains (D), where 10 mM and 15 mM A23187 generally reduced
enzyme activities. (E) Cathepsin B/L activity was higher at the 3 h time point. 5 mM and 10 mM induced cathepsin B/L activity
and 15 mM reduced cathepsin B/L activity. Values are expressed relative to CTRL (vehicle-treated) 3 h, which has been arbitrarily
given a value of 1.0. *po0.05, signiﬁcantly different than 3 h (main effect of time). Main effects (po0.05) for A23187 dose are
indicated with superscript letters, where groups with different letters are signiﬁcantly different from each other.
D. Bloemberg, J. Quadrilatero / Data in Brief 7 (2016) 1024–10301028A23187 experiments). For CisPL experiments, cisplatin was dissolved fresh in sterile saline (0.9% NaCl)
at 3 mM, diluted in warmed culture media to working concentrations of 25 mM, 50 mM, or 100 mM,
and administered to cells for 8 h, 16 h, or 24 h (Fig. 1). A23187 was dissolved in DMSO at a con-
centration of 1 mM, stored at 20 °C, and diluted in warmed culture media to working concentra-
tions of 5 mM, 10 mM, or 15 mM before being administered to cells for 3 h or 6 h (Fig. 1).
2.2. Preparation of cell lysates
At the indicated time points, cells were collected via trypsinization, centrifuged at 1000g, and
stored at 80 °C. For enzyme activity assays, cell lysates were generated by sonicating cells in lysis
buffer containing 20 mM HEPES, 10 mM NaCl, 1.5 mM MgCl, 1 mM DTT, 20% glycerol, and 0.1% Triton-
X100 at a pH of 7.4. Separate cells were prepared for immunoblotting using lysis buffer containing
protease inhibitors (Complete Cocktail, Roche). Protein content of cell lysates was determined using
the BCA protein assay method.
2.3. Proteolytic enzyme activity
Enzymatic activity of caspase-3, 8, 9, cathepsin B/L, and calpain was measured with the
ﬂuorogenic substrates Ac-DEVD-AMC, Ac-IETD-AMC, Ac-LEHD-AMC, Z-FR-AFC, and Suc-LLVY-AMC (Enzo
Life Sciences), respectively [5,6]. Calpain activity was determined by accounting for the reduction in
ﬂuorescence caused by the calpain inhibitor Z-LL-CHO (Enzo Life Sciences). Cell lysates were incubated in
duplicate in black 96-well plates with the appropriate substrate at room temperature, 30 °C, or 37 °C for
caspase, cathepsin, and calpain analyses, respectively. Fluorescence was measured using a SPECTRAmax
Gemini XS microplate spectroﬂuorometer. Enzyme activity was normalized to total protein content and
expressed as ﬂuorescence intensity in arbitrary units (AU) per milligram protein.
2.4. Immunoblotting
Immunoblotting was performed as previously described [6,9,10]. Samples were loaded with equal
amounts of protein (30 mg) and separated by SDS/PAGE before being transferred onto PVDF membranes.
Membranes were probed with primary antibodies against Bcl2 (1:200), Bax (1:1000), PARP (1:500),
Fig. 5. Changes to expression of apoptotic signaling proteins in response to A23187. A23187 treatments had concentration-
dependent effects on the Bax/Bcl2 ratio (A), p53 (B), and pH2AX (C). (D) Representative immunoblots. (E) Brightﬁeld micro-
scope images of Giemsa stained CTRL (vehicle-treated) and A23187 treated cells. Values are expressed relative to CTRL 3 h,
which has been arbitrarily given a value of 1.0. Main effects (po0.05) for A23187 dose are indicated with superscript letters,
where groups with different letters are signiﬁcantly different from each other. Scale bar represents 100 mm.
D. Bloemberg, J. Quadrilatero / Data in Brief 7 (2016) 1024–1030 1029pH2AX (1:500), p53 (1:500) (Santa Cruz Biotechnology), cleaved caspase-3 (1:1000), or actin (1:2000)
(Sigma Aldrich) overnight at 4 °C and then incubated with the appropriate horseradish peroxidase
(HRP)-conjugated secondary antibody (Santa Cruz Biotechnology) for 1 h at room temperature. Bands
were visualized using the Clarity ECL blotting substrate (Bio-Rad) and the ChemiGenius 2 Bio-Imaging
System (Syngene). The approximate molecular weight for each protein was estimated using Precision
Plus Protein WesternC Standards and Precision Protein Strep-Tactin HRP Conjugate (Bio-Rad).2.5. Microscopy
Giemsa staining was performed to assess changes to cell morphology [11]. Brieﬂy, cells were ﬁxed
in ice-cold methanol for 10 min, allowed to air-dry, and incubated with 1:20 Giemsa solution (Sigma
Aldrich) for 45 min at room temperature. Images were acquired using a Nikon microscope equipped
with a PixeLink digital camera.
D. Bloemberg, J. Quadrilatero / Data in Brief 7 (2016) 1024–103010302.6. Statistics
Results are presented as means7SEM, where n¼3. For comparisons across time points, 2-way
ANOVAs were performed with Tukey post-hoc analyses to detect main effect differences. For com-
parisons at individual time points in Figs 3, 1-way ANOVAs were performed with Tukey post-hoc
analyses to detect differences between treatment groups. In all cases, po0.05 was considered sta-
tistically signiﬁcant.Acknowledgments
This research was supported by funds (341256) provided by the Natural Sciences and Engineering
Research Council of Canada (NSERC) to Joe Quadrilatero. Darin Bloemberg is the recipient of a NSERC
postgraduate scholarship. NSERC did not participate in study design, the collection, analysis and
interpretation of data, the writing of the report, or the decision to submit the article for publication.Appendix A. Supplementary material
Supplementary data associated with this article can be found in the online version at http://dx.doi.
org/10.1016/j.dib.2016.03.032.References
[1] P. Saikumar, Z. Dong, V. Mikhailov, M. Denton, J.M. Weinberg, M.A. Venkatachalam, Apoptosis: deﬁnition, mechanisms, and
relevance to disease, Am. J. Med. 107 (1999) 489–506.
[2] G. Kroemer, L. Galluzzi, C. Brenner, Mitochondrial membrane permeabilization in cell death, Physiol. Rev. 87 (2007) 99–163.
[3] X. Wan, L.J. Helman, Effect of insulin-like growth factor II on protecting myoblast cells against cisplatin-induced apoptosis
through p70 S6 kinase pathway, Neoplasia 4 (2002) 400–408.
[4] S. Salucci, M. Battistelli, S. Burattini, C. Squillace, B. Canonico, P. Gobbi, S. Papa, E. Falcieri, C2C12 myoblast sensitivity to
different apoptotic chemical triggers, Micron 41 (2010) 966–973.
[5] T.L. Campbell, A.S. Mitchell, E.M. McMillan, D. Bloemberg, D. Pavlov, I. Messa, J.G. Mielke, J. Quadrilatero, High-fat feeding
does not induce an autophagic or apoptotic phenotype in female rat skeletal muscle, Exp. Biol. Med. 240 (2015) 657–668.
[6] D. Bloemberg, J. Quadrilatero, Mitochondrial pro-apoptotic indices do not precede the transient caspase activation asso-
ciated with myogenesis, Biochim. Biophys. Acta 1843 (2014) 2926–2936.
[7] I. Kim, W. Xu, J.C. Reed, Cell death and endoplasmic reticulum stress: disease relevance and therapeutic opportunities, Nat.
Rev. Drug Discov. 7 (2008) 1013–1030.
[8] D. Malinska, B. Kulawiak, A. Wrzosek, W.S. Kunz, A. Szewczyk, The cytoprotective action of the potassium channel opener
BMS-191095 in C2C12 myoblasts is related to the modulation of calcium homeostasis, Cell. Physiol. Biochem. 26 (2010)
235–246.
[9] E.M. McMillan, J. Quadrilatero, Autophagy is required and protects against apoptosis during myoblast differentiation,
Biochem. J. 462 (2014) 267–277.
[10] A.S. Mitchell, I.C. Smith, D. Gamu, S. Donath, A.R. Tupling, J. Quadrilatero, Functional, morphological, and apoptotic
alterations in skeletal muscle of ARC deﬁcient mice, Apoptosis 20 (2015) 310–326.
[11] A. Fanzani, A. Zanola, F. Rovetta, S. Rossi, M.F. Aleo, Cisplatin triggers atrophy of skeletal C2C12 myotubes via impairment of
Akt signalling pathway and subsequent increment activity of proteasome and autophagy systems, Toxicol. Appl. Phar-
macol. 250 (2011) 312–321.


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Caspase activity and apoptotic signaling in proliferating C2C12 cells following cisplatin or A23187 exposure

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