Piscidin-1 Induces Apoptosis via Mitochondrial Reactive Oxygen Species-Regulated Mitochondrial Dysfunction in Human Osteosarcoma Cells

Osteosarcoma (OSA) is the most common type of cancer that originates in the bone and usually occurs in young children. OSA patients were treated with neoadjuvant chemotherapy and surgery, and the results were disappointing. Marine antimicrobial peptides (AMPs) have been the focus of antibiotic research because they are resistant to pathogen infection. Piscidin-1 is an AMP from the hybrid striped bass (Morone saxatilis × M. chrysops) and has approximately 22 amino acids. Research has shown that piscidin-1 can inhibit bacterial infections and has antinociception and anti-cancer properties; however, the regulatory effects of piscidin-1 on mitochondrial dysfunction in cancer cells are still unknown. We aimed to identify the effects of piscidin-1 on mitochondrial reactive oxygen species (mtROS) and apoptosis in OSA cells. Our analyses indicated that piscidin-1 has more cytotoxic effects against OSA cells than against lung and ovarian cancer cells; however, it has no effect on non-cancer cells. Piscidin-1 induces apoptosis in OSA cells, regulates mtROS, reduces mitochondrial antioxidant manganese superoxide dismutase and mitochondrial transmembrane potential, and decreases adenosine 5′-triphosphate production, thus leading to mitochondrial dysfunction and apoptosis. The mitochondrial antioxidant, mitoTempo, reduces the apoptosis induced by piscidin-1. Results suggest that piscidin-1 has potential for use in OSA treatment.


Influence of Piscidin-1 on OSA cells apoptosis.
To further elucidate the link between the OSA cell apoptosis induced by piscidin-1, we conducted an in vitro proof-of-principle study. First, we used annexin V-FITC (green) and propidium iodide (PI)-red staining with flow cytometry dot-plot diagrams to analyze the apoptosis induced by piscidin-1. Figure 2A shows the typical shift between apoptotic cells (annexin V+/PI−) and dead cells (annexin V+/PI+) from the left quadrant toward the right quadrant in MG63 cells treated with piscidin-1 for 24 h. At 5 and 10 μM piscidin-1, the rates of apoptotic and dead MG63 cells (14.48 ± 4.38% and 45.59 ± 3.61%, respectively) obviously increased compared with those in the control (4.61 ± 0.16%, 0 μM piscidin-1) (Fig. 2B), whereas a similar phenomenon can also be observed in piscidin-1-treated 143 B cells (Fig. S2B,C). Furthermore, a terminal deoxy-nucleotidyl transferase dUTP nick end labeling (TUNEL) assay was conducted in vitro to evaluate the apoptotic effect of piscidin-1 and observe the apoptotic cells that exhibited extensive DNA fragmentation during apoptosis 29 . TUNEL staining (green) was exhibited using immunofluorescence and showed nuclear condensation and apoptotic bodies in the MG63 cells after treatment with 10 μM piscidin-1, and all nuclei (blue) were stained with 4′,6-diamidino-2-phenylindole (DAPI) (Fig. 2C). Our data show that there was an increasing trend in TUNEL-positive cells in the groups treated with 5 (19.48 ± 4.38%) and 10 μM (40.59 ± 3.60%) piscidin-1 compared with that in the control (5.11 ± 1.39%, 0 μM piscidin-1) (Fig. 2D). The intrinsic apoptosis pathway is initiated by the disruption of the inner mitochondrial membrane under excessive oxidative stress, thus resulting in the release of cytochrome c (cyt c) protein 30 , whereas the downstream caspase-3 has been identified as an important executioner of the apoptosis process 31 . After normalization of the protein with β-actin, analysis revealed that the exposure of MG63 cells to different concentrations of piscidin-1 for 24 h increased the expression levels of cytosolic cyt c, cleaved caspase-9, and cleaved caspase-3 (Fig. 2E). After protein normalization with cyt c oxidase complex IV (COX IV), the mitochondrial cyt c was not affected. MG63 cells that were treated with different concentrations of piscidin-1 (i.e., 0, 1, 5, and 10 μM) for 24 h exhibited a rapid accumulate in cytoplasmic cyt c protein levels of 1.00 ± 0.33, 15.89 ± 1.93, 18.06 ± 1.50, and 18.20 ± 5.00 in a dose-dependent manner, but mitochondrial cyt c was not affected (Fig. 2F). The piscidin-1 treatment of the MG63 cells with 1, 5, and 10 μM obviously increased the protein levels of cleaved caspase-9 in a dose-dependent manner to 3.18 ± 0.50, 4.76 ± 0.73, and 5.67 ± 0.86, respectively, compared with that of the control at 1.00 ± 0.17 (0 μM piscidin-1). The piscidin-1 treatment of the MG63 cells with 1, 5, and 10 μM also led to a dose-dependent increase in the levels of which were treated with 0, 0.1, 1, 5, or 10 μM piscidin-1 for 24 h and assayed using MTT staining to measure cell viability. Piscidin-1 did not obviously influence the cell viability of two noncancer cell lines. (D) Human primary gingival fibroblast cells (HGF-1) cells and (E) oral mucosal fibroblast (OMF) cells were treated with the 0, 0.1, 1, 5, or 10 μM piscidin-1 for 24 h and assayed using MTT to measure the changes in cell viability. Cell viability is expressed as a percentage of the untreated control cells (0 μM). The results are expressed as the mean ± SEM of three independent experiments. Significance was determined using Student's t-test. *p < 0.05; **p < 0.01. (F) MG63 cells were treated with 0, 0.1, and 10 μM piscidin-1 for 24 h, and images were taken with a scanning electron microscope. The photograph represented 200X (upper) and 400X (lower) magnification.
www.nature.com/scientificreports www.nature.com/scientificreports/ cleaved caspase-3 at 11.6121 ± 6.17, 16.52 ± 2.92, and 28.02 ± 4.62, respectively, compared with that of the control at 1.00 ± 0.43 (0 μM piscidin-1) (Fig. 2G). These observations indicated that piscidin-1-induced apoptosis in OSA cells is through the release of cyt c from the mitochondria and the subsequent activation of caspase-9 and caspase-3. Whole cell lysate proteins were loaded for Western blot analysis by using cleaved caspase-9, cleaved caspase-3, cyt c, and β-actin. β-actin and COX IV were used as the cytosol and mitochondria internal controls, respectively. The groupings were cropped from different gels subjected to identical conditions. Full blots are shown in the Supplementary Information, Fig. S4. Cytosol cytochrome c, mitochondrial cytochrome c (F), and protein levels of cleaved caspase-9 and cleaved caspase-3 (G) were quantified and normalized to that of β-actin and COX IV and were expressed as fold changes. Significance was determined using Student's t-test. *p < 0.05; **p < 0.01.
Pretreatment with mtROS scavengers rescue the mtROS increase, MTP dissipation, and apoptosis induced by Piscidin-1. mitoTempo is a mitochondria-targeting antioxidant and an mtROS scavenger, i.e., it protects the mitochondria from oxidative damage 36 . To determine the effect of mitoTempo on mtROS overproduction, MTP dissipation, and apoptosis induced by piscidin-1, MG63 cells were pretreated with or without 5 μM mitoTempo for 2 h. Thereafter, 10 μM piscidin-1 was added and allowed to react for 24 h. The fluorescence intensity of MitoSOX Red showed a 7.38 ± 0.59-fold change in the mitoTempo and piscidin-1 cotreatment group and a 16.09 ± 1.24-fold change in the piscidin-1-only treatment group compared with that in the dimethyl sulfoxide (DMSO) control group, thus indicating that cotreatment with mitoTempo substantially reduced mtROS production in MG63 cells treated with piscidin-1 (Fig. 7A). The piscidin-1 (10 μM) treatment of MG63 cells for 24 h induced MTP dissipation, but the pretreatment of the cells with mitoTempo effectively rescued this dissipation from an MTP of 44.69 ± 3.87% to an MTP of 95.72 ± 4.67% (Fig. 7B). To evaluate whether mitoTempo attenuates the cell apoptosis induced by piscidin-1, MG63 cells were treated with piscidin-1, mitoTempo, or both for 24 h. The proportion of dead cells (upper right) and apoptotic cells (lower right) were calculated using the gated , and β-actin (as an internal control). The groupings were cropped from different gels subjected to identical conditions. Full blots are shown in the Supplementary  Information, Fig. S6. The complex I subunit (B), complex II subunit (C), complex III subunit (D), complex IV subunit (E), and complex V subunit (F) protein levels were quantified and normalized to the β-actin level. (G) The effects of the total ATP production in MG63 cells treated with various concentrations of piscidin-1 for 24 h were measured using an ATP fluorescence kit. The results are expressed as the mean ± SEM of three independent experiments. Significance was determined using Student's t-test; *p < 0.05; **p < 0.01. (2020) 10:5045 | https://doi.org/10.1038/s41598-020-61876-5 www.nature.com/scientificreports www.nature.com/scientificreports/ quadrant dot-plot profile (Fig. 7C). The fold change was 0.99 ± 0.09 in the group treated with both mitoTempo and piscidin-1 and was 3.23 ± 0.22 in the group treated with only piscidin-1 compared with that in the DMSO vehicle control, thus suggesting that there was a considerable reduction in cell apoptosis from cotreatment with mitoTempo (Fig. 7D). Our data indicate that piscidin-1 treatment increased mtROS production, disrupted MTP integrity, and promoted cell apoptosis, whereas pretreatment with mitoTempo sharply suppressed mtROS and cell apoptosis and reduced MTP dissipation.

Discussion
Piscidin-1 peptide, which was generously donated by the laboratory of Professor Jyh-Yih Chen at Academia Sinica, Jiaushi, Ilan, Taiwan, is one of the AMP series and natural marine compounds isolated from the mast cells of the hybrid striped bass (Morone saxatilis × M. chrysops). Piscidin-1 is composed of approximately 22 amino acid peptides with amphiphilic α-helical conformation structure and has a small molecular weight of ~2.5 kDa. Previous studies have shown that piscidin-1 can inhibit bacterial proliferation 27 , induce Hela and fibrosarcoma cell apoptosis 28 , and relieve pain 37 . Our results demonstrate that piscidin-1 obviously reduces the cell viability of MG63 cells (IC 50 = 6.72 ± 2.31 µM) and 143 cells (IC 50 = 7.15 ± 1.55 µM) compared with that in A549 cells (IC 50 = 9.93 ± 1.25 µM) and SKOV3 cells (IC 50 = 13.56 ± 1.84 µM); however, piscidin-1 has no effect on HGF-1 or OMF. The process of apoptosis is generally identified by distinct morphological characteristics (including DNA strand breaks, externalization of phosphatidylserine residues on the cell surface, cell shrinkage, chromosome www.nature.com/scientificreports www.nature.com/scientificreports/ condensation, and cell apoptotic body formation) and is an energy-dependent mechanism 36 . Most natural marine compounds with anticancer properties have complex action mechanisms, among which the regulations of the apoptosis induction and mitochondrial dysfunction signaling networks play key roles 38 . First, we observed the morphological changes in OSA cells treated with piscidin-1 by using scanning electron microscopy, and the results showed that the structure of the cell cytoskeleton became spherical at low concentrations of the peptide. High concentrations of the peptide induced cell shrinkage, which represents cell apoptosis. The additional examinations using flow cytometric annexin V/PI staining, TUNEL fluorescence assay, and Western blot analysis using antibodies against apoptosis factors (cyt c, cleaved caspase-9, and cleaved caspase-3) confirmed that piscidin-1 induces apoptosis. The current study demonstrated that piscidin-1 treatment inhibits OSA cell growth at low concentrations, with an IC 50 of ~6-7 µM. Lin et al. reported that piscidin-1 induces cell death in HeLa and HT1080 cells with an IC 50 of ~7-8 µM 28 , similar to that in our results. Furthermore, our experiments show that the antitumor activity of piscidin-1 in OSA cells was initiated by inducing apoptosis, and this finding is consistent with the results of a previous report 28 .
MtROS are harmful substances and are the undesirable byproducts of oxidative phosphorylation, which indefinitely damages cells; however, superoxide anion (O 2 − ) and hydrogen peroxide (H 2 O 2 ) are major ROS and important signaling molecules and are directly involved in the regulation of mitochondrial and cellular functions 39 . Considering that SOD2 enzymes can extensively catalyze undue ROS in the mitochondria, they are involved in ROS production and in changes in the respiratory chain complex process 40 . Apoptosis induction uses two main pathways, namely, extrinsic (death receptor) and intrinsic (mitochondria mediated) 41,42 . The intrinsic pathway regulates the permeability of the mitochondrial outer membrane and creates pores in this membrane that cause apoptosis-inducing factors, such as cyt c, to be constantly released into the cytoplasm. Cyt c and caspase-9 combine to form an apoptosome and subsequently activate caspase-3, thus causing apoptosis and death. It is www.nature.com/scientificreports www.nature.com/scientificreports/ worth noting that the accumulation of mtROS precedes MTP (ΔΨm) damage, nuclear condensation, and apoptotic body formation 43 . This study demonstrated that the effective cytotoxicity of and the apoptosis induced by piscidin-1 on OSA cells is achieved by the induction of mtROS and the disruption of MTP; however, we also observed that piscidin-1 increases mtROS levels at very low concentrations (0.1 µM) before the observed MTP dissipation (1 µM).
The mitochondria plays many important roles in eukaryotic cells, the most important of which is the production of ATP during the OXPHOS process 44 . There are two ways that cells produce ATP: OXPHOS (in the mitochondria) and non-mitochondrial (in the cytoplasm) processes. Our study showed that piscidin-1 reduces non-mitochondrial and OXPHOS respiration, including the basal OCR, ATP production, maximum respiration OCR, spare respiration capacity, and non-mitochondrial respiration but has no effect on proton leak respiration in OSA cells. The inner membrane of the mitochondria has many folds (cristae), which accommodate many copies of the respiratory chain component or OXPHOS complexes I-V. Complexes I-V is multi-subunit enzymes that synergistically produce an electrochemical proton gradient on the inner mitochondrial membrane. Finally, together with complex V (ATP synthase), they form the machinery for producing ATP, which is the source of cell metabolism and cell biosynthesis and is also known as the energy currency of cells 45 . The expression levels of complex I-V proteins were gradually reduced with increased concentrations of piscidin-1. The cell mitochondrial stress test using a Seahorse XF24 Analyzer and the expressions of complex I-V proteins indicated that the mitochondria exhibited a two-stage change as the piscidin-1 concentration increased. First, it was stimulated and damaged, efficiency was slightly reduced, and it maintained stable operations. Second, if the damage was too extensive, it would not be able to work appropriately, and the entire cell would be on the verge of collapse. This indicated that piscidin-1 has obvious apoptosis effects on OSA cells by inhibiting mitochondrial OXPHOS and the expression of complexes I-V, thus decreasing ATP production and finally causing mitochondrial dysfunction.
Mitochondria are dynamic organelles that exhibit fusion (combining pieces) and fission (splitting into smaller pieces) via the active recruitment of specific proteins to the indicated locations within the organelle. Mitochondrial fusion requires three large GTPases, the outer membrane proteins MFN1 and MFN2 and the inner membrane protein OPA1. The activation of GTPase DRP1 and the outer membrane protein FIS1 is necessary for mitochondrial fission. Mitochondrial fusion and fission have several important functions. They control the morphology of the mitochondria and allow for the exchange of content between the mitochondria, thus controlling mitochondrial distribution and promoting the release of membrane gap proteins during apoptosis 46 . Several structural changes in the mitochondria are important for rapid and efficient apoptosis they must have a fragmented expression with the permeable outer membrane, and the cristae must be disconnected; therefore, mitochondrial fission has significant implications in oxidative stress response and apoptosis 47 . Our results are consistent with the aforementioned observation, i.e., the induction of apoptosis by piscidin-1 is responsible for a decrease in the mitochondrial fusion proteins and an increase in the mitochondrial fission proteins in OSA cells.
MitoTempo is a mitochondria-targeted antioxidant with superoxide scavenging properties, i.e., it helps protect against oxidative damage to the mitochondria 48 . Many researches have proved that mitoTempo maintains mitochondrial integrity and reduces necrosis and apoptosis 48,49 . Our results demonstrated that piscidin-1 induces OSA cell apoptosis by increasing mtROS production and destroying MTP. Pretreatment with mitoTempo significantly decreases the overproduction of mitochondrial superoxide radical, the disruption of MTP, and the apoptosis mediated by perscidin-1 in the OSA cells.
Most of the defense peptides with anticancer activity are cationic and the molecular structures are either α-helical or β-sheet. Compared with normal cells, the cellular membrane of cancer cells contains more anions such as heparin sulfate and phospholipid phosphatidylserine, so that the cell membrane exhibits net negative charge. During the early stage of apoptosis, the mitochondria gradually become negatively charged with the increase of cardiolipin exposure; therefore, piscidin-1 entering OSA cells will attack and destroy the integrity of the mitochondria 50 . Current studies suggest that the selectivity and underlying mechanism of these anti-cancer peptides depends on invasion of mitochondrial and/or plasma membranes of cancer cells by potential differences, leading to cell apoptosis 51 . Piscidin-1 with positive charge possesses an α-helical structure, and the specific anticancer ability we observed may be caused by the aforementioned mechanism 28 .
The results of this study showed that piscidin-1, which is a marine pepide, inhibits MG63 and 143B OSA cell growth by inducing the apoptosis pathway (Fig. 8). Piscidin-1 treatment enhances mtROS production and decreases SOD2 antioxidant expression, followed by disrupting OXPHOS, decreasing the expression of complex I-V proteins, inducing an imbalance in mitochondrial dynamic proteins (including increased fission proteins and decreased fusion proteins), dissipating MTP (Δψm) and ATP production, and causing cancer cell apoptosis. MitoTempo is effective in reducing the apoptosis mediated by piscidin-1. These results indicate that piscidin-1 possesses anticancer effects by increasing mtROS and inducing mitochondrial dysfunction. To the best of our knowledge, this is the first report to demonstrate that piscidin-1 can inhibit human cancer growth by influencing mtROS, OXPHOS, and mitochondrial function. The above characteristics indicate that piscidin-1 has the potential for additional research and development as a new chemotherapeutic drug. Scanning electron microscopy morphological analysis. The scanning electron microscopy sample was prepared on the basis of the methods of Kung et al. 52 . The cell cultures were washed with PBS and subjected to dehydration in a graded alcohol series for 2 min each (10%, 25%, 50%, 75%, and absolute ethanol v/v % ethanol). After dehydration, the samples were mounted on copper plates and sputter-coated with a thin layer of gold. Finally, the scanning electron microscope images were obtained using the JEOL JSM-7000F scanning electron microscope at an accelerating voltage of 5 kV.

Methods
Cell viability. The cells were seeded at an initial density of 6 × 10 3 cell/well in 96-well plates and placed on a culture plate into a CO 2 incubator at 37 °C overnight. After piscidin-1 treatment with 0, 0.1, 1, 5, and 10 µM for 24, 48, and 72 h, 20 µL 5 mg/mL MTT solution was added into the cell-attached wells for 4 h. The supernatant was removed and intracellular formazan compounds were solubilized in 100 µL/well DMSO at room temperature for 15 min. The absorbance at a wavelength of 570 nm was measured using an enzyme-linked immunosorbent assay microplate reader (Dynatech Laboratories, Chantilly, VA, USA). The relative cell viability was calculated as the percentage of cells treated with piscidin-1 and those in the untreated control group.
Flow cytometric analyses for apoptosis. Apoptosis was measure by fluorescence levels after staining with annexin V-FITC and PI. The seeded 5 × 10 5 cells were plated in six-well plates and incubated overnight. MG63 and 143B cells were treated with piscidin-1 at 0, 0.1, 1, 5, and 10 μM for 24 h, another group was treated with or without mitoTempo for 2 h, and 10 μM piscidin-1 was added and allowed to react for 24 h. The resuspended cells at 1 × 10 5 cells/100 μL in working binding buffer (HBSS) containing 5 μL purified recombinant annexin V-FITC and 5 μL PI were gently mixed and incubated for 10 min at room temperature to avoid light, and 1 mL HBSS solution was added. Beckman Coulter's CytoFLEX (Southfield, MI, USA) was used to detect the fluorescence intensity of annexin V (green fluorescence)/PI (red fluorescence). At least 20,000 cells/group were analyzed using CytExpert 2.0 software (Beckman Coulter). TUNEL staining and fluorescence image. For the TUNEL assay purchased from Roche, 1 × 10 5 cells/ well were set on glass coverslips for each well in a 12-well plate for at least 24 h and were subsequently treated with piscidin-1 for 24 h. The cell culture medium was carefully removed and washed twice with PBS, and the MG63 cells were fixed and blocked with a 4% neutral formalin and 3% BSA solution for 15 min. The staining procedure followed the manufacturer's instructions. The cell nuclei were stained with DAPI as a nuclear position. Fluorescence was visualized using the Leica TCS SP5 II confocal microscope (Wetzlar, Germany).

Flow cytometric analyses for mtROS and MTP.
The fluorescence levels of mtROS were measured after staining with 10 μM MitoSOX Red (red fluorescence) for 20 min at 37 °C. MTP was indicated using rhodamine 123 (green fluorescence) and flow cytometry in the model of FITC according to the manufacturer's instructions. The cells were seeded at a density of 5 × 10 5 cells/well in a six-well plate. After treatment with various concentrations of piscidin-1, mitoTempo, or cotreatment of piscidin-1 and mitoTempo, the fluorescent dyes were added at the proper volumes into the HBSS solution, and the solution was incubated at 37 °C for 20 min. Subsequently, the medium was removed, and trypsin was added to the cells. The cells were then resuspended in 1 mL HBSS. The CytoFLEX (Beckman Counter) was used to detect the fluorescence intensity of mtROS and MTP. At least 20,000 cells/group were analyzed using CytExpert 2.0 software (Beckman Coulter).
Mitochondrial and cytosol isolation assay. The MG63 cells were treated with 0, 0.1, 1, 5, and 10 μM piscidin-1 for 24 h, and the mitochondria and cytosol were separated and isolated using the Mitochondria/ Cytosol Fractionation kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's instructions. Thereafter, Western blot analysis was conducted.
Western blot analysis. The MG63 cells were seeded at a density of 1 × 10 6 cells/well in a 6 cm dish and were pretreated with 0, 0.1, 1, 5, and 10 μM piscidin-1 for 24 h. RIPA buffer (Thermo Fisher Scientific, USA) was used on the cells to isolate total protein, and the lysates were centrifuged at 13,000 rpm at 4 °C for 30 min to obtain soluble proteins in the supernatant. A BCA assay was then used to determine protein concentrations (Bio-Rad, Hercules, CA, USA). Extracts of protein were taken from each group and then fractionated using 8-15% sodium dodecyl sulfate-polyacrylamide gel electrophoresis to separate the proteins. Proteins were transferred to a polyvinylidene difluoride (PVDF) membrane (Millipore, Bedford, MA, USA). Antibodies for cyt c, COX IV, SOD2, cleaved caspase-3 and cleaved caspase -9 (Cell Signaling Technology, Danvers, MA, USA), β-actin, mitochondrial complex protein (complexes I, II, III, IV, and V), and mitochondrial dynamic-related protein (MFN1, MFN2, OPA1, DRP1, and FIS1) (Abcam, Cambridge, UK) were used. The membranes were incubated at 37 °C for 1 h with horseradish peroxidase-conjugated secondary antibodies. The membrane was placed on a visualization strip by using a chemiluminescence kit (Millipore, Darmstadt, Germany) and UVP BioChemi imaging (UVP LLC, Upland, CA, USA). The PVDF membrane was reprobed using a β-actin antibody as a loading control. The relative densitometry of the bands was quantified using ImageJ, normalized with that of the β-actin and COX IV levels, and expressed as fold changes. ATP concentration assay. MG63 cells were seeded in triplicate at a density of 2 × 10 5 cells/well in six-well plates and then treated with various concentrations of piscidin-1 for 24 h. The cells were harvested in a lysis buffer (20 mM glycine, 50 mM MgSO4, and 4 mM ethylenediaminetetraacetic acid) 53 . ATP was measured using the ATP Colorimetric/Fluorometric Assay kit (BioVision, Inc., Milpitas, CA, USA) according to the manufacturer's instructions. Twenty microliters from each sample were mixed with 4 μL ATP solution for fluorescence readings at Ex/Em 535/587 nm by using a fluorescence meter. The amount of ATP production was determined from a standard curve constructed with 10-100 pmol ATP.
Live-cell metabolic assay. OCR was measured using the XF24 Seahorse XF Analyzer (Seahorse Bioscience, North Billerica, MA, USA). MG63 and 143B cells (30,000/well) were cultured onto XF24 polystyrene cell culture plates and then treated with piscidin-1 for 24 h. The old medium was removed, and 675 μL of DMEM medium that was adjusted to pH 7.4 without sodium bicarbonate was added before using the XF24 Analyzer. For three injection stages, 1 μM oligomycin, 0.5 μM FCCP, and 1 μM antimycin A/rotenone were sequentially added into the assay medium. The attached cells were lysed using RIPA, and a BCA kit was used to measure the optical density at 570 nm to normalize OCR value.
Statistical analyses. SPSS ver. 17 (SPSS Inc., Chicago, IL, USA) was used to analyze the data. The variables analyzed using the independent Student's t-test were expressed as mean ± standard error. *p < 0.05 and **p < 0.01 were considered statistically significant analyses.

Data availability
All data generated or analyzed during this study are included in the published article (and its Supplementary Information file).