Introduction. Jeong A Lim 1,2, Sung Ho Hwang 1, Min Jeong Kim 1, Sang Soo Kim 2 and Hye Sun Kim 1

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1 N-terminal cleavage fragment of focal adhesion kinase is required to activate the survival signalling pathway in cultured myoblasts under oxidative stress Jeong A Lim 1,2, Sung Ho Hwang 1, Min Jeong Kim 1, Sang Soo Kim 2 and Hye Sun Kim 1 1 Department of Biological Science, College of Natural Sciences, Ajou University, Suwon, Korea 2 Radiation Medicine Branch, National Cancer Center, Goyang, Korea Keywords calpain; focal adhesion kinase; myoblasts; oxidative stress; survival signalling Correspondence H. S. Kim, Department of Biological Science, College of Natural Sciences, Ajou University, Suwon , Korea Fax: Tel: hsunkim@ajou.ac.kr (Received 11 April 2012, revised 16 June 2012, accepted 9 July 2012) doi: /j x We have previously shown that the cultured L6 myoblasts are susceptible to menadione-induced oxidative stress. Damaged cells were detached from the culture dishes. In the present study, we focused on focal adhesion kinase (FAK), which plays pivotal roles in maintaining focal adhesion function and cell survival. FAK, normally localized at the focal adhesion regions of the myoblasts, was not observed at the regions under oxidative stress induced by menadione and H 2 O 2. Two cleavage products, 80-kDa N-terminal FAK and 35-kDa C-terminal FAK fragments, as well as fulllength FAK (125 kda) were detected in myoblasts cultured under normal conditions by western blotting with anti-n-terminal FAK or anti-c-terminal FAK sera. Of interest was the finding that the cleavage products of FAK (but not full-length FAK) disappeared under oxidative stress. The cleavage of full-length FAK to N-terminal FAK and C-terminal FAK was inhibited by calpeptin, a specific calpain inhibitor. In addition, pre-incubation of cells with calpeptin resulted in a sharp decrease in survival signals, such as Akt phosphorylation and the ratio of Bcl-2 Bax, under stress conditions. By contrast, not only relative viability, but also Akt phosphorylation and the ratio of Bcl-2 Bax was significantly improved when cells were transfected with a DNA construct of N-terminal FAK-Myc. These results suggest that the N-terminal FAK positively regulates survival signalling in early phases of oxidative stress in the cultured myoblasts. Introduction Reactive oxygen species (ROS) such as superoxide anions, hydrogen peroxide and hydroxyl radicals are generated through various intracellular pathways in several types of cells. Low concentrations of ROS can stimulate signalling pathways that regulate cell proliferation, migration and differentiation [1]. However, a high concentration and accumulation of ROS results in macromolecular damage, including lipid peroxidation, protein oxidation and DNA oxidation [2]. Disturbance in the balance between free radical production and the effectiveness of defence systems can increase the free radical concentration, leading to oxidative stress. The skeletal muscle is a highly oxygenated tissue and normally exposed to ROS; therefore, skeletal muscle cells are susceptible to damage by ROS [3]. During muscle contraction, the oxygen flux and energy supply in skeletal muscle cells change rapidly. It has been estimated that 3 5% of the total electron flux results in ROS formation [4]. The high concentration of myoglobin in skeletal muscle is an additional factor Abbreviations, C-terminal FAK; FAK, focal adhesion kinase; FERM, protein 4.1, ezrin, radixin and moesin homology; FRNK, FAK-related nonkinase;, N-terminal FAK; PI3K, phosphoinositide 3-kinase; ROS, reactive oxygen species; WST, water-soluble tetrazolium salt. FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3573

2 is required to activate survival signalling J. A. Lim et al. contributing to oxidative damage. A transient protein radical that induces oxidative damage to biomolecules is produced during the reaction between myoglobin and hydrogen peroxide [5]. Cells respond to oxidative stress by activating survival signalling pathways. In a previous study, we showed that phosphoinositide 3-kinase (PI3K) Akt signalling is responsible for the differential susceptibility of myoblasts and myotubes to menadione-induced oxidative stress [6]. Menadione (2-methyl-1,4-naphthoquinone), also known as vitamin K 3, is a quinone compound that induces oxidative stress and apoptosis in cultured myoblasts [6,7]. We also showed that p85, the regulatory subunit of PI3K, can interact with phosphorylated focal adhesion kinase (FAK) in cultured myoblasts [8]. FAK is a nonreceptor tyrosine kinase localized at focal adhesions and plays a key role in cell migration [9,10], cell differentiation [11,12] and antiapoptotic signalling pathways [13]. Under hydrogen peroxide-induced oxidative stress, FAK acts as an upstream signalling molecule in the PI3K Akt pathway and promotes cell survival against stress [14]. Previous studies showed that hydrogen peroxide causes tyrosine phosphorylation of FAK, as well as serine phosphorylation of Akt [14,15]. FAK is composed of a protein 4.1, ezrin, radixin and moesin homology (FERM) domain, a central kinase domain, two proline-rich regions and the C-terminal focal adhesion targeting domain. Binding of the FERM domain to integrin receptors induces autophosphorylation of FAK at Tyr397 [16], which provides binding sites for the SH2 domains of Src and p85 [17]. Recently, calpain-mediated proteolysis of FAK was reported to regulate adhesion dynamics in motile cells [18]. The site where FAK is cleaved by calpain lies after the Ser745 located between the two C-terminal proline-rich regions. Calpains are calcium-dependent cysteine proteases that have been proposed to regulate migration, proliferation, differentiation and apoptosis in various cell types [19 21]. There are two ubiquitous calpain isoforms: calpain 1 (l-calpain) requires calcium concentrations in the micromolar range for activation and calpain 2 (m-calpain) requires calcium concentrations in the millimolar range [22]. Both calpain 1 and calpain 2 are involved in cleavage of focal adhesion proteins, including FAK in vitro [23]. However, calpain 2 (but not calpain 1) activity is required for cleavage of focal adhesion proteins such as FAK, paxillin, spectrin and talin in fibroblasts [22]. Myoblasts detach from a culture dish under oxidative stress, indicating that focal adhesion molecules are damaged by stress. In the present study, we investigated the activity of FAK under menadione-induced oxidative stress. Although FAK is involved in survival signalling, little is known regarding its precise function under oxidative stress. We observed that three types of FAK were present under normal culture conditions: full-length FAK (125 kda), 80-kDa N-terminal () fragment and 35-kDa C-terminal () fragment. Under menadione-induced oxidative stress, disappearance of the cleaved forms of FAK (but not full-length FAK) was observed. Survival signals such as Akt signalling and the Bcl-2 Bax ratio increased under menadione-induced oxidative stress when the -Myc construct was transfected into myoblasts. These results suggest that is involved in the Akt survival signalling pathway in cultured myoblasts under menadione-induced oxidative stress. Results Disappearance of the cleavage products of FAK under oxidative stress The skeletal muscle produces a complex set of ROS at rest as well as during contractile activity. The muscle tissue precisely regulates these ROS and prevents potentially deleterious effects [24]. Despite this regulation, a high concentration of ROS can damage cells. In a previous study, we showed that cultured myoblasts such as L6 and C2 cells were damaged by both menadione and H 2 O 2, which induce oxidative stress [6]. In the present study, we examined the morphological changes in cultured myoblasts under oxidative stress. Damaged cells showed altered shapes (round form) and were detached from the bottom of the dish (Fig. 1A). This observation suggests that oxidative stress inhibits focal adhesion. Therefore, we focused on FAK, an important focal adhesion molecule that plays pivotal roles in maintaining focal adhesion and in cell survival [13,16]. To investigate FAK localization, L6 myoblasts were exposed to menadione or H 2 O 2 for 1 h and then immunostained using anti-fak serum. FAK was present in the focal adhesion regions in the control cells (Fig. 1B, arrowheads). However, FAK was not observed in focal adhesion regions after menadione or H 2 O 2 exposure (Fig. 1B). Next, we investigated whether the amount of FAK changes with varying levels of menadione exposure. Myoblasts were exposed to increasing doses of menadione for 1 h, and the FAK level was determined by performing western blotting with anti- and anti- sera to detect full-length FAK as well as fragmented FAK. As shown in Fig. 2, three major types of FAK were detected in cultured myoblasts that had not been exposed to menadione (lane 0): 3574 FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

3 J. A. Lim et al. is required to activate survival signalling A Control Menadione H 2 O 2 Bar = 50 µm B FAK DAPI Phase Merge Fig. 1. Disappearance of FAK from the focal adhesion region under oxidative stress. (A) L6 myoblasts were exposed to 20 lm menadione or 1 mm H 2 O 2 for 2 h and then observed under a phase contrast microscope. Arrows indicate the cells detached from the substratum of a culture dish. (B) Myoblasts grown on coverslips were exposed to menadione or H 2 O 2 for 1 h. Cells were incubated with anti-fak serum and stained using tetramethylrhodamine-5- isothiocyanate-conjugated anti-mouse IgG antibody. 4,6-Diamidino-2-phenylindole was used to identify the nucleus. Arrowheads indicate FAK in the focal adhesion region. Control Menadione H 2 O 2 Bar = 10 µm full-length FAK (125 kda), (80 kda), and C- FAK (35 kda). However, with increasing doses of menadione, levels of and decreased in a dose-dependent manner (Fig. 2A), whereas the fulllength FAK slightly increased. In a previous study, we showed that apoptosis occurred with menadione at a concentration of 20 lm in L6 myoblasts [6], in which the concentrations of both and almost disappeared. To confirm the disappearance of FAK under oxidative stress, we exposed the cells to H 2 O 2. Similar to the results obtained with menadione exposure, the levels of and (but not fulllength FAK) significantly decreased after H 2 O 2 exposure (Fig. 2B). In addition to the three bands, other bands are also detected by anti-fak sera. FAK can be cleaved by caspases as well as calpain and the bands could be the products of caspases. We observed a similar response to oxidative stress and the cleavage pattern of FAK in C2 cells (Fig. S1). Calpain is responsible for the cleavage of FAK A recent study showed that FAK is cleaved by calpain during adipocyte differentiation [12] but not under oxidative stress. Therefore, we attempted to identify the protease that cleaves FAK into and under normal culture conditions for myoblasts. To determine the protease(s) involved in FAK cleavage, C2 myoblasts were incubated with several protease inhibitors, PSI (a proteasome inhibitor), Z-VAD (a pancaspase inhibitor) or calpeptin (a specific calpain inhibitor) containing DMEM for 1 h, and the pattern of FAK cleavage was determined. The proteasome is known to play a role under various stress conditions [25], and caspases were previously reported to be implicated in FAK cleavage [26]. However, neither PSI nor Z-VAD affected the pattern of FAK cleavage. By contrast, calpeptin blocked FAK cleavage. In calpeptin-treated cells, both and were only minimally detected (Fig. 3A). To confirm whether calpain cleaves the full-length FAK into and, L6 myoblasts were exposed to increasing doses of calpeptin and incubated for 1 h. Disappearance of FAK fragments was observed in the presence of 20 lm calpeptin, and almost no fragments were observed at concentrations > 50 lm (Fig. 3B). Calpeptin is a reversible and cell-permeable peptide aldehyde inhibitor of calpain [27,28]. Therefore, we anticipated that and would reappear when calpeptin was removed from the culture media. After incubation with calpeptin for 1 h, neither nor was detected (Fig. 3C, lane 1). However, these fragments reappeared 1 h after FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3575

4 is required to activate survival signalling J. A. Lim et al. A IB: IB: B Menadione (µm) (kda) H 2 O (mm) (enhanced) Fig. 2. Disappearance of FAK cleavage products under oxidative stress. L6 myoblasts were exposed to menadione (A) or H 2 O 2 (B) for 1 h at the indicated doses. The amount of FAK was detected using specific anti- or anti- sera. For better resolution, the intensity of the band was increased by a longer exposure (enhanced). was used as a loading control. removal of the agent (Fig. 3C, lane 2). These results show that calpain is responsible for cleaving the fulllength FAK into and in cultured myoblasts. Myoblasts are more vulnerable to oxidative stress when calpain is blocked by calpeptin Next, we blocked calpain activity and examined the response of the cells under menadione-induced oxidative stress. C2 myoblasts pre-incubated with calpeptin or dimethysulfoxide for 1 h were exposed to menadione for an additional 1 h. In dimethysulfoxide-preincubated cells, both and were detected without menadione exposure. However, these products disappeared after incubation with menadione for 1 h (Fig. 4A). By contrast, the fragments were not detected in calpeptin-pre-incubated cells, regardless of menadione exposure. These results correspond to previous results shown in Fig. 3B. When FAK is activated and autophosphorylated at Tyr397, it binds the SH2 domain of PI3K and subsequently activates PI3K [29]. Because contains the Tyr397 phosphorylation site, we investigated whether is also phosphorylated at Tyr397 by performing western blotting with anti-phospho-fak (Tyr397) serum. As shown in Fig. 4A, as well as full-length FAK was phosphorylated at Tyr397. However, after incubation with menadione, phosphorylation was not observed. This result suggests that could modulate downstream signalling pathways such as the Src and PI3K Akt signalling pathways. Among the FAK-related survival signalling pathways, we examined the expression and phosphorylation of Akt, which is a downstream effector of PI3K [14]. The Akt expression level was not affected by exposure to menadione, regardless of calpeptin preincubation. However, in dimethysulfoxide-pre-incubated cells, Akt phosphorylation (Ser473) increased with menadione exposure with minimal changes in protein level. By contrast, the increase in phosphorylation was not prominent in calpeptin-pre-incubated cells. Under the same culture conditions, the expression of both Bcl-2, an anti-apoptotic molecule, and Bax, a pro-apoptotic molecule, was assayed. Bcl-2 neutralizes the pro-apoptotic effects by forming heterodimers with Bax. Thus, a decrease in the Bcl-2 Bax ratio indicates an increased level of apoptosis [30]. After calpeptin-pre-incubation, Bcl-2 expression was reduced, whereas that of Bax was increased. The ratio of Bcl-2 to Bax shown is the mean of three independent experiments (Fig. 4B). These results suggest that the cells are more vulnerable to menadione-induced oxidative stress when calpain is blocked by calpeptin (Fig. 4B). We also assayed cell viability using the water-soluble tetrazolium salt (WST) assay (Fig. S2). Analysis of relative cell viability showed that L6 cells pre-incubated with calpeptin were more vulnerable to the effects of menadione than cells pre-incubated with dimethysulfoxide. After 6 or 9 h of incubation, the relative viability of calpeptin-pre-incubated cells was approximately half that of dimethysulfoxide-pre-incubated cells (Fig. S2). Overexpression of in myoblasts increased resistance to oxidative stress We assumed that the cleavage products of FAK are involved in survival signalling under menadioneinduced oxidative stress. To validate our hypothesis, we evaluated the possible role of the fragment containing Tyr397, which may be related to survival signalling. A Myc-tagged construct was 3576 FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

5 J. A. Lim et al. is required to activate survival signalling A B Control DMSO PSI Calpeptin Z-VAD Calpeptin Full-length FAK Fig. 3. Calpeptin blocks the cleavage of full-length FAK. (A) C2 myoblasts were incubated for 1 h with the protease inhibitors: 1 lm PSI (a proteasome inhibitor), 20 lm Z-VAD (a pan-caspase inhibitor) or 50 lm calpeptin (a specific calpain inhibitor). Next, the cells were harvested and the FAK level was determined using western blotting with anti- or anti- sera. Dimethysulfoxide was used as a vehicle. (B) Cells were exposed to the indicated doses of calpeptin for 1 h, and FAK was detected using anti- or anti- sera. (C) Cells incubated with 50 lm calpeptin for 1 h (lane 1) were separated from the agent and then incubated for an additional 1 h in fresh, serum-free DMEM (lane 2). The cells were harvested, and FAK was detected using anti- or anti- sera. was used as a loading control (µm) C 1 2 DMEM Calpeptin Calpeptin removal menadione exposure. The relative viability of overexpressing cells was 20% more than that of control vector-transfected cells (Fig. 5A). After 6 h of menadione incubation, the cells were harvested and overexpression was determined using anti-myc serum. As shown in Fig. 5B, overexpression was detected with both anti-myc and anti- sera. Western blotting using anti-phospho-fak serum showed that the overexpressed was phosporylated. The level of decreased with menadione exposure in control vectortransfected cells. Levels of overexpressed also slightly decreased after menadione exposure, although this may be a consequence of the decrease in endogenous after menadione exposure. By contrast, the levels of full-length FAK also slightly increased with the overexpression of. Interestingly, overexpression dramatically induced Akt phosphorylation without changing Akt protein levels. In addition, the increased ratio of Bcl-2 Bax in cells overexpressing after menadione exposure (Fig. 4C) corresponded to the increased ratio of cell viability (Fig. 4A). These results suggest that, a product of calpain cleavage, is involved in survival signalling via Akt phosphorylation after menadioneinduced oxidative stress in cultured myoblasts. Discussion 1 h Harvest (Lane 2) 0 h Harvest (Lane 1) generated as described in the Materials and methods. C2 cells were then transfected with control vector (pcdna 3.1 myc-his LacZ) or Myc-tagged and exposed to menadione. The results of the WST assay indicated that both type of cells were damaged by menadione exposure. However, cells overexpressing were shown to be resistant to the effects of In the present study, we showed that the fragment, a product of calpain cleavage, is responsible for Akt-involved survival signalling in the early phases of oxidative stress conditions. FAK is known to play an important role in survival signalling pathways; therefore, we focused on the role of FAK fragments under oxidative stress. and, which are products of calpain activity, as well as full-length FAK, were observed in myoblasts cultured under normal conditions. Interestingly, the cleavage products (but not FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3577

6 is required to activate survival signalling J. A. Lim et al. A Menadione B Bcl-2/Bax ratio Calpeptin Menadione DMSO + Calpeptin p - p - p -Akt Akt Bcl-2 Bax DMSO Calpeptin Fig. 4. Myoblasts incubated with calpeptin are more susceptible to oxidative stress. (A) C2 myoblasts pre-incubated with dimethysulfoxide or 50 lm calpeptin for 1 h were exposed to 20 lm menadione for an additional 1 h. The cells were harvested, and the FAK level was determined using anti- or anti- sera. Concurrently, phosphorylation was determined using anti-phospho-fak (Tyr397). The levels of Akt, phospho-akt (Ser473), Bcl-2 and Bax were determined by western blotting with the specific antibodies. was used as a loading control. (B) The expression of Bcl-2 and Bax was determined using SCION IMAGE (Scion Corp., Fredrick, MD, USA). Bcl-2 and Bax levels at the indicated times are expressed as a ratio. Values represent the mean ± SEM of three independent experiments (*P < 0.05). full-length FAK) disappeared under oxidative stress. Using calpeptin, a calpain inhibitor, as well as overexpression of, we showed that the cleavage products play an important role in survival signalling in cultured myoblasts under oxidative stress. The present study was based on the observation that cultured myoblasts detach from the bottom of the culture dish under oxidative stress conditions (Fig. 1). When bound to fibronectin, the a5b1 integrin receptor triggers a survival signal in many tissue culture cell lines [31]. In the absence of the a5b1 integrin fibronectin interaction, many cell types undergo anoikis (i.e. apoptotic cell death that occurs in * nontransformed cells when their substratum-cell adhesions are disrupted) [32,33]. Cultured myoblasts are also known to undergo anoikis when surface modifications lead to changes in the functional status of the a5b1 integrin receptor [34]. FAK, a key mediator of integrin signalling, has been shown to play an important role in survival of anchorage-dependent cells [35]. Recently, FAK cleavage was reported in various cell types under conditions such as c-myc-induced apoptosis (chicken embryonic fibroblasts) [36], growth factor deprivation-induced apoptosis (human umbilical vein endothelial cells) [26], cell migration [18] and adipocyte differentiation [12]. FAK is a caspase substrate present in cells undergoing apoptosis [26,37]. In anoikis of intestinal epithelial cells, FAK cleavage by caspase-3 and caspase-6 has been reported [38]. Moreover, FAK is proposed to be a critical target of oxidative stress-activated caspases, which, in turn, accelerate subsequent apoptotic cell death, regardless of the anchorage status of cells [37]. Our results showed no decrease in the levels of full-length FAK during the early stress period, although levels of fulllength FAK eventually decreased with longer stress (Fig. S1); this corroborates the results obtained in previous studies [26,37,39]. Accordingly, we examined the possibility that caspase is involved in FAK degradation in cultured myoblasts. We found an additional cleavage product of FAK, which was smaller than, when L6 myoblasts were cultured under serum-deprived conditions (Fig. S3). The smaller cleavage product disappeared after treatment with Z-VAD-FMK, a pan-caspase inhibitor (Fig. S3). The caspase cleavage site is Asp722 on the C-terminal domain of FAK [40], whereas the calpain cleavage site is Ser745 [18]. Therefore, the size of N-terminal fragment after caspase cleavage may be smaller than the produced by calpain. This suggests that the smaller fragment is a product of caspase cleavage. However, the smaller fragment was not observed under oxidative stress conditions, as well as under normal culture conditions. Hence, we excluded the possibility that caspase(s) are involved in FAK degradation in myoblasts under oxidative stress under these culture conditions. Another point worthy of note is that may be FAK-related nonkinase (FRNK), which is known to act as an endogenous FAK inhibitor. FRNK is expressed as an independent protein and consists of the C-terminal domain of FAK and the proline-rich region and focal adhesion targeting domain [41]. FRNK negatively regulates the proliferation, differentiation and migration of vascular smooth muscle cells [42,43]. The size of the observed in the present 3578 FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

7 J. A. Lim et al. is required to activate survival signalling A Relative viability (%) B C Bcl-2/Bax ratio Mena * * + + study was similar to that of FRNK. However, in the previous study, FRNK was not detected in skeletal muscle tissue or L6 myoblasts [43]. Thus, we excluded the possibility that was FRNK. By contrast, several studies report possible functions for the N-terminal domain of FAK. For example, this fragment is known to induce rounding, detachment and apoptosis * Control vector Incubation (h) Control vector Control vector Mena + * LacZ-Myc -Myc p - p - p -Akt Akt Bcl-2 Bax Fig. 5. Phosphorylation of Akt increases after overexpression. (A) C2 myoblasts were transfected with the control vector (pcdna3.1 myc-his LacZ) or the -pcdna3.1 myc-his using Lipofectamine2000Ô. Next, the cells were exposed to 20 lm menadione for the indicated times. Cell viability was measured using the WST assay, and relative viability is expressed as the ratio of each treatment with respect to that just before menadione incubation (0 h) for the control vector-transfected cells. Values are mean ± SEM of three separate experiments. The asterisks indicate that the viability of -transfected cells was significantly different from that of control vector-transfected cells (*P < 0.05). (B) Under the same culture conditions, cells were harvested after 6 h of menadione exposure, and the expression of the transfected protein was detected using a specific Myc-tag antibody. The amounts of other proteins were determined by western blotting with antibodies specific to the indicated proteins. was used as a loading control. (C) Bcl-2 Bax is the level after dimethysulfoxide treatment in control vector-transfected cells. Values are expressed as the mean ± SEM of three independent experiments (*P < 0.05). in breast carcinoma cells but not in nontransformed cells [44]. In addition, the N-terminal domain of FAK interacts directly with p53 and inhibits p53 transcriptional activity. This interaction consequently leads to cell survival signalling [45]. The FERM domain of FAK, which is also located on the N-terminal region, mediates the nuclear translocation of FAK; this translocation is important for p53 inhibition [46]. Therefore, we also examined whether FAK in the focal adhesion region moves to the nuclear region under oxidative stress. Myoblasts cultured under oxidative stress or without stress were harvested, and nuclear fractions were separated to obtain the cytosol fraction. Western blotting was performed with marker proteins, with calpain in the cytosol fraction and histones in the nuclear fraction, and nuclear localization of or fulllength FAK was not observed under oxidative stress (Fig. S4). Hence, it is unlikely that plays a role in the nuclear region under this oxidative condition. The cleavage of FAK by calpain is reported to be important in adipocyte differentiation [12]. Calpain activity also plays an important role in myoblast differentiation [47,48]. Therefore, we examined whether the cleavage of FAK by calpain is related to the progress of myoblast differentiation. Indeed, the level of increased with differentiation progression in L6 myoblasts (Fig. S5). In a previous study, we showed that, compared to myotubes, myoblasts are more vulnerable to oxidative stress, and this differential susceptibility is dependent on the PI3K Akt signalling pathway [6]. Taken together, we suggest that, which is increased by calpain activity during differentiation, may be involved in the PI3K Akt signalling pathway, which enhances resistance to FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3579

8 is required to activate survival signalling J. A. Lim et al. oxidative stress. In a previous study, we also showed that phosphorylated FAK interacts with PI3K during myoblast differentiation [8]. The phosphorylation of FAK at Tyr397 is important for FAK activation and downstream signalling [29]. Because also contains this site, we focused on the role of in myoblasts under oxidative stress. The observation that the overexpression of increased the phosphorylation of Akt and the Bcl-2 Bax ratio under oxidative stress (Fig. 5) suggests that is not only a product of calpain under oxidative stress conditions, but also induces survival signalling. In conclusion, we have shown that products of FAK cleavage by calpain activity are required for activating survival signalling in cultured myoblasts under oxidative stress conditions. How these cleavage products regulate the survival signalling pathway remains to be determined. However, we showed that overexpression of induces Akt phosphorylation and increases the Bcl-2 Bax ratio. These results suggest that positively regulates the Akt signalling pathway in myoblasts under oxidative stress, resulting in the resistance to oxidative stress. Materials and methods Materials Cell culture materials, including fetal bovine serum, horse serum and DMEM, were obtained from Hyclone (Logan, UT, USA). pcdnaô 3.1 myc-his vector and Lipofectamine2000Ô were purchased from Invitrogen (Carlsbad, CA, USA). T4 DNA ligase and NotI restriction enzyme were obtained from Roche (Mannheim, Germany). BamHI restriction enzyme was obtained from Fermentas (Hanover, MD, USA). Taq polymerase was from New England Biolabs (Ipswich, MA, USA). H 2 O 2 and calpeptin were purchased from Tocris Cookson (Ellisville, MO, USA). Anti- (clone 4.47) and anti- sera were purchased from Upstate Biotechnology (Lake Placid, NY, USA). Anti-phospho-FAK (Tyr397) serum was obtained from Abcam (Cambridge, UK). Anti-phospho-Akt (Ser473), anti-akt and anti-bcl-2 sera were obtained from Cell Signaling Technologies (Danvers, MA, USA). Anti- Bax and anti-myc tag sera were obtained from Millipore (Billerica, MA, USA). Anti-actin serum was obtained from Santa Cruz (Santa Cruz, CA, USA). The WST assay reagent (EZCytox) was purchased from IT Bio (Seoul, Korea). Menadione, protease inhibitor cocktail and other reagents were obtained from Sigma (St Louis, MO, USA). Cell culture and induction of oxidative stress L6 and C2 myoblasts were cultured as described previously [8]. Briefly, myoblasts were seeded at a concentration of cellsæml )1 in a 100-mm culture dish. The cells were grown in DMEM supplemented with 10% fetal bovine serum in humidified air containing 5% CO 2 at 37 C. The cells were split every third day. To induce oxidative stress, myoblasts were pre-incubated in serum-free DMEM for 1 h and then exposed to menadione or H 2 O 2. Dimethysulfoxide was used as a vehicle. Immunostaining L6 myoblasts cultured on coverslips coated with poly l-lysine were incubated with menadione or dimethysulfoxide. The cells were fixed with 4% paraformaldehyde in NaCl P i for 10 min and permeabilized using 0.5% Triton X-100 in NaCl P i. After blocking with 1% BSA in NaCl P i for 20 min, the cells were incubated with anti- sera (dilution 1 : 100) overnight at 4 C. The cells were incubated with a tetramethylrhodamine-5-isothiocyanate-conjugated secondary antibody (dilution 1 : 100) for 1 h. To stain the nuclei, cells were incubated with 4,6-diamidino-2- phenylindole. The cells were observed using a confocal microscope (LSM510; Zeiss, Jena, Germany). Viability assay Cell viability was determined using the WST assay. Cells exposed to menadione were grown in 96-well plates, and WST reagent was added to each well. Plates were incubated for 2 h at 37 C. A 455 of solubilized formazan was measured using an ELISA plate reader (Bio-Rad, Hercules, CA, USA). Preparation of cell extracts and western blotting At varying culture times, cells were washed three times with ice-cold NaCl P i and harvested using a rubber scraper. Cells were disrupted in lysis buffer (20 mm Hepes, ph 7.4, 250 mm sucrose, 2 mm ethylene glycol tetraacetic acid, 5 mm NaN 3, 0.1 mm Na 3 VO 4, 10 mm NaF and 0.1% protease inhibitor cocktail) using ultrasonication. Subsequently, the cells were centrifuged at 1000 g for 10 min at 4 C to remove cell debris. Protein concentrations were determined by the Bradford assay using Bio- Rad dye reagent with BSA as a standard. Equal amounts of proteins were separated using SDS PAGE, and the separated proteins were transferred to a poly(vinylidene fluoride) membrane. The membrane was blocked with 4% nonfat milk or BSA in 10 mm Tris (ph 7.5), 100 mm NaCl and 0.1% Tween-20 for 1 h, and then incubated with the indicated primary antibodies for 1 h at room temperature or overnight at 4 C. After incubation with a peroxidase-conjugated secondary antibody for 1 h at room temperature, immunoreactive protein bands were visualized using the enhanced chemiluminescence method FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS

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11 J. A. Lim et al. is required to activate survival signalling 48 Louis M, Zanou N, Van Schoor M & Gailly P (2008) TRPC1 regulates skeletal myoblast migration and differentiation. J Cell Sci 121, Supporting information The following supplementary material is available: Fig. S1. disappears in C2 under oxidative stress. Fig. S2. Calpeptin decreases the viability under oxidative stress. Fig. S3. is not relevant to caspases. Fig. S4. does not exist in the nucleus. Fig. S5. increases during differentiation. This supplementary material can be found in the online version of this article. Please note: As a service to our authors and readers, this journal provides supporting information supplied by the authors. Such materials are peer-reviewed and may be reorganized for online delivery, but are not copy-edited or typeset. Technical support issues arising from supporting information (other than missing files) should be addressed to the authors. FEBS Journal 279 (2012) ª 2012 The Authors Journal compilation ª 2012 FEBS 3583