Reactive oxygen species (ROS) are harmful for the human body, and exposure to ultraviolet irradiation triggers ROS generation. Previous studies have demonstrated that ROS decrease mitochondrial membrane potential (MMP) and that Mg2+ protects mitochondria from oxidative stress. Therefore, we visualized the spatio-temporal dynamics of Mg2+ in keratinocytes (a skin component) in response to H2O2 (a type of ROS) and found that it increased cytosolic Mg2+ levels. H2O2-induced responses in both Mg2+ and ATP were larger in keratinocytes derived from adults than in keratinocytes derived from newborns, and inhibition of mitochondrial ATP synthesis enhanced the H2O2-induced Mg2+ response, indicating that a major source of Mg2+ was dissociation from ATP. Simultaneous imaging of Mg2+ and MMP revealed that larger Mg2+ responses corresponded to lower decreases in MMP in response to H2O2. Moreover, Mg2+ supplementation attenuated H2O2-induced cell death. These suggest the potential of Mg2+ as an active ingredient to protect skin from oxidative stress.
Reactive oxygen species (ROS) are constantly produced in the human body and have harmful effects. Exposure to ultraviolet (UV) irradiation in particular is a notable trigger for ROS generation1. ROS generation is attributed to several factors represented by enzyme activities of the electron transport chain in mitochondria2. Oxidative stress induced by ROS constitutes a harmful condition because ROS have high reactivity and cause DNA damage, lipid peroxidation, and protein carbonylation. These forms of oxidative damage increase with age3,4. Excessive ROS generation also causes mitochondrial dysfunction. In cellular-level experiments, the addition of H2O2 (a type of ROS) decreased mitochondrial membrane potential (MMP)5,6. Moreover, it has been reported that ROS-induced mitochondrial dysfunction contributes to various diseases, including Alzheimer’s disease, type 1 diabetes, atherosclerosis, and cancer7. Skin in particular is frequently exposed to ROS stress since UV irradiation from sunlight is the main generator of ROS1; ROS stress has been suggested to be involved in aging, inflammation, and pathogenesis of skin cancer, among other conditions8,9. Therefore, protecting biomolecules and mitochondria from ROS is critical for maintaining normal cellular functions, and cells express antioxidants and several mechanisms to avoid oxidative stress10. Skin cells, such as the keratinocytes and fibroblasts which constitute the epidermis, also have ROS clearance mechanisms11. In our previous study, we suggested that Mg2+ was involved in mechanisms to help cells avoid oxidative stress12.
Mg2+ is the most abundant divalent cation in living cells and is related to more than 600 enzymes as a cofactor13,14,15. Recent studies have demonstrated that small changes in intracellular Mg2+ can have large impacts on cellular events, such as cell division, maturation of neurons, and neurodegeneration16,17,18. In neurons and cancer cells, intracellular Mg2+ is stored in the mitochondria and constitutes Mg2+ an important factor for sustainable mitochondrial functioning12,19. The impact of Mg2+ in relation to retaining MMP has been reported at the cellular and isolated mitochondrial levels20,21,22. The protective effects of Mg2+ toward oxidative stress have also been reported in various types of cells, such as endothelial cells23,24, bone marrow mesenchymal stem cells25,26, and chick embryo hepatocytes27. On the other hand, in monocytes the environment is the determining factor in whether high levels of Mg2+ will increase or decrease ROS levels28. These findings clearly indicate the important role of Mg2+ in protecting cells from oxidative stress. However, it has not been well understood whether cells change intracellular Mg2+ concentrations in response to oxidative stress, nor exactly how Mg2+ protects cells from oxidative stress.
In this study, we examined changes in Mg2+ concentration in response to ROS in keratinocytes. Keratinocytes were chosen because they are one of the most ROS-exposed cells in the human body, as ROS are generated by UV from sunlight. The dynamics of cytosolic Mg2+ were visualized using fluorescence imaging under H2O2 stress to find that H2O2 induced an increase in cytosolic Mg2+ concentration ([Mg2+]cyto) due to the release of Mg2+ bound to ATP upon the H2O2-induced decrease in ATP level. The effects of Mg2+ on mitochondrial functions were investigated by comparison to changes in MMP to find that the increased Mg2+ attenuated the decrease in MMP. Interestingly, Mg2+ supplementation further suppressed the decrease in MMP and H2O2-induced cell death. Our results suggest that Mg2+ provides robustness to intracellular ATP levels under oxidative stress.
H2O2-induced cytosolic Mg2+ increase
Spatio-temporal dynamics of Mg2+ in keratinocytes derived from adults (adult keratinocytes) were visualized with an Mg2+ selective indicator, KMG-104. It was confirmed that H2O2 did not directly react with KMG-104 (Supplementary Fig. 1a). H2O2 caused the [Mg2+]cyto to increase in keratinocytes from 40-year-old donor (Fig. 1a–c). The average time-course of [Mg2+]cyto increased immediately after application of 1 mM H2O2 and reached a plateau at 5 min (Fig. 1a). The spatial distribution of the changes in [Mg2+]cyto was almost uniform within the cells, whereas the amplitude of the change in each cell varied (Fig. 1c). To examine whether the H2O2-induced Mg2+ increase constituted a common phenomenon in keratinocytes across individual differences and age, the responses were compared in five keratinocyte cell lines: three from newborns (three different 0 years old donors: newborn keratinocytes) and two from adults (40 and 57 years old donors: adult keratinocytes). There was large variability in the amplitudes of the H2O2-induced Mg2+ response in each cell, regardless of age (Fig. 1d and Supplementary Fig. 2a). While some newborn keratinocytes increased and others decreased in [Mg2+]cyto in response to H2O2 for all three cell lines, most of the adult keratinocytes showed increases in [Mg2+]cyto. As a result, all newborn keratinocyte lines showed no or slightly decreased [Mg2+]cyto responses on average, whereas adult keratinocytes lines exhibited increased [Mg2+]cyto on average (Supplementary Fig. 2b). Therefore, the data were divided into two groups, newborn and adult, and the difference in these two groups were compared. The amplitude of H2O2-induced Mg2+ responses was significantly larger in adult keratinocytes than in newborn keratinocytes (Fig. 1e). A higher concentration of H2O2 (10 mM) elicited increases in [Mg2+]cyto, even in newborn keratinocytes, indicating that this is not a phenomenon specific to adult keratinocytes, but simply that adult keratinocytes are more sensitive to H2O2 than newborn keratinocytes (Supplementary Fig. 2c, d). The responses to 10 mM H2O2 were also larger in adult keratinocytes than in newborn keratinocytes (Supplementary Fig. 2e, f). To examine underlying mechanism of the increase in [Mg2+]cyto and role of the Mg2+, the following experiments were performed in adult keratinocytes from a 40-year-old donor.
A major source of Mg2+ is Mg2+ dissociation from ATP in the process of ATP consumption
To identify the Mg2+ source that was responding to H2O2, we examined Mg2+ entry from an extracellular medium, and fluorescence imaging was performed in the medium without Mg2+. An H2O2-induced Mg2+ increase was still observed in this condition (Fig. 2a), which indicates that H2O2 induced the release of Mg2+ from an intracellular Mg2+ source. In other cell types, mitochondria were identified as intracellular Mg2+ storage sites and released Mg2+ into cytosol upon a depolarization of MMP19,29,30. Simultaneous imaging of [Mg2+]cyto and MMP revealed that FCCP, an uncoupler of mitochondria, rapidly decreased MMP; however, FCCP did not increase [Mg2+]cyto in keratinocytes, but rather deceased it (Fig. 2b). This indicates that depolarization of the mitochondria did not cause Mg2+ release from mitochondria in keratinocytes.
Next, the involvement of Mg2+ transporters was investigated. It has been reported that Na+/Mg2+ exchangers, one of which is SLC41A1, mediate Mg2+ efflux from the cells and are inhibited by quinidine14,22,31. Therefore, if one of the targets of H2O2 is the Na+/Mg2+ exchanger, which leads to the increase in [Mg2+]cyto, it is expected that quinidine also increases [Mg2+]cyto and that prior application of quinidine abolishes the H2O2-induced increase in [Mg2+]cyto. Quinidine alone induced increase in [Mg2+]cyto in both normal and Mg2+-free conditions, whereas vehicle (DMSO, final concentration 0.5%) decreased [Mg2+]cyto, indicating that inhibition of Mg2+ efflux leads to increase in [Mg2+]cyto in keratinocytes (Supplementary Fig. 3a). The amplitude of quinidine-induced Mg2+ increase was greater in normal condition than Mg2+-free condition (Supplementary Fig. 3b). These results suggest that [Mg2+]cyto is normally balanced by Mg2+ influx and efflux. The effect of quinidine on H2O2-induced Mg2+ responses was also investigated. The responses were greater in the presence of quinidine than that in the presence of vehicle both in normal and Mg2+-free medium (Fig. 2c, d), while those were slightly smaller in the presence of vehicle (DMSO, final concentration 0.5%) than in the absence of vehicle (compare Fig. 2a and c). This result indicates that the Na+/Mg2+ exchanger does not mediate H2O2-induced Mg2+ responses and that inhibition of Mg2+ efflux retains Mg2+ released from intracellular Mg2+ sources.
Most of the intracellular Mg2+ binds to various biomolecules, and a major binding partner of Mg2+ in the cytoplasm is ATP. ATP normally binds to Mg2+ in the form of an Mg–ATP complex, but the Mg2+ is dissociated when ATP is consumed and degraded to ADP, leading to an increase in free Mg2+. Mg2+ dissociation from ATP due to ATP consumption has been reported as the cause of increases in [Mg2+]cyto during mitosis and apoptosis18,32. To confirm the dissociation of Mg2+ from ATP, the genetically encoded ATP sensor ATeam33 was expressed in keratinocytes and the intracellular ATP level was visualized (Fig. 3a). It was confirmed that H2O2 does not directly affect ATeam signals independently of ATP (Supplementary Fig. 1b). Upon an application of H2O2, ATP concentration decreased in keratinocytes (Fig. 3b blue line). To examine the relationship between the decrease in ATP and increase in [Mg2+]cyto, H2O2-induced change in ATP concentration was also examined in the newborn keratinocytes. These cells showed smaller increases in H2O2-induced [Mg2+]cyto (Fig. 1e) and relatively smaller decreases in ATP compared to adult keratinocytes (Fig. 3b red line and c), whereas there were no significant difference in cellular ATP contents between those cells (Supplementary Fig. 4). We also examined whether the cells with large ATP decreases showed larger increases in [Mg2+]cyto. Mitochondria are a major source of cellular ATP, and their inhibition affects cellular ATP production. Therefore, the effects of prior inhibition of ATP production in the mitochondria on the H2O2-induced responses in ATP and Mg2+ were investigated. Neither oligomycin, which is an inhibitor of FoF1 ATP synthase, nor FCCP alone elicited decreases in ATP levels at least within minutes of the application (Supplementary Fig. 5a, b). The efficacy of oligomycin and FCCP was confirmed by the results that when combined with the glycolysis inhibitor 2-deoxy-d-glucose (2DG), those induced greater decreases in cellular ATP levels than 2DG alone (Supplementary Fig. 5c, d). These results also indicate that mitochondria and glycolysis complement each other to maintain ATP concentration in keratinocytes. Interestingly, pretreatment with oligomycin or FCCP significantly enhanced H2O2-induced decreases in ATP (Fig. 3d, e). The inhibition of ATP synthesis in the mitochondria also enhanced the H2O2-induced increase in [Mg2+]cyto, although neither oligomycin alone nor FCCP alone induced increases in [Mg2+]cyto (Fig. 3f, g), and Mg2+ and ATP dynamics in response to H2O2 were mirror images of each other (compare Fig. 3d and f). These results indicate that Mg2+ dissociation from ATP was the major Mg2+ source in response to H2O2 in keratinocytes.
Mg2+ suppresses the H2O2-induced decrease in MMP in a concentration-dependent manner
Our next question was whether increased [Mg2+]cyto protected cells from oxidative stress and via what mechanism this occurred. It has been reported that H2O2 suppresses mitochondrial function by decreasing MMP5,6. On the other hand, previous studies show that Mg2+ contributes to the maintenance of MMP and that increased Mg2+ by Mg2+ supplementation or inhibition of Mg2+ efflux attenuates decreases in MMP under cellular stress conditions20,21,22. Therefore, the relationship between [Mg2+]cyto and MMP was examined using simultaneous imaging with KMG-104 and TMRE (Fig. 4a). H2O2 caused an increase in [Mg2+]cyto and a gradual decrease in MMP within 10 min (Fig. 4b). Keratinocytes that showed large Mg2+ increases exhibited lower decreases in MMP (thin solid lines in Fig. 4b), and conversely, cells with small increases in [Mg2+]cyto showed large decreases in MMP (thin dotted lines in Fig. 4b). To prove that Mg2+ had a direct effect on H2O2-induced decreases in MMP, the cytosolic level of Mg2+ was increased by adding Mg2+ to the extracellular medium before H2O2 stimulation. Supplementation of Mg2+ to the extracellular medium increased the Mg2+ concentration in the medium from 0.9 to 5 mM and led to a steep increase in [Mg2+]cyto. The subsequent application of H2O2 did not induce significant changes in averaged [Mg2+]cyto, while some cells showed an increase or decrease in [Mg2+]cyto (green line in Fig. 4c). Interestingly, H2O2-induced decreases in MMP were significantly prevented by the prior addition of Mg2+ (Fig. 4d, e), whereas Mg2+ supplementation itself had little effect on MMP (red line in Fig. 4c). These findings suggest that [Mg2+]cyto affects MMP primarily under stress conditions. To estimate the relationship between [Mg2+]cyto and H2O2-induced decreases in MMP, the changes in [Mg2+]cyto from initial levels (shown as green lines in Fig. 4b, c) and the H2O2-induced changes in MMP that were normalized at 1 min before H2O2 application (from the data in Fig. 4d) in each cell were plotted (Fig. 4f). In both the normal condition (gray dots) and the high Mg2+ condition (orange dots), strong correlations between Mg2+ increases and MMP were observed; higher levels of cytosolic Mg2+ were correlated with lower decreases in MMP. Interestingly, these two plots appear to line up on the same line, indicating that [Mg2+]cyto is one of the key determinants for protecting the mitochondria from H2O2 damage (Fig. 4f).
Finally, we confirmed that the Mg2+ supplementation suppresses the toxicity of H2O2. Exposure to 1 mM H2O2 for 24 h induced ~40% cell death in keratinocytes in a normal culture medium. Supplementation of additional 5 mM Mg2+ to the culture medium suppressed the toxicity (Fig. 5). Our results indicate that Mg2+ supplementation is effective in protecting keratinocytes from H2O2 toxicity.
The skin is continuously exposed to oxidative stress. This study revealed that H2O2, which is a kind of ROS, causes increases in [Mg2+]cyto in human keratinocytes. This H2O2-induced Mg2+ response was higher in adult keratinocytes than in newborn keratinocytes. Our findings indicate that the source of Mg2+ was dissociation from ATP in the process of ATP consumption. Upon the addition of H2O2, decreases in MMP were also observed, and the change in MMP was strongly correlated with increases in [Mg2+]cyto. In other words, higher levels of cytosolic Mg2+ were connected to lower MMP decreases. Furthermore, H2O2-induced decreases in MMP were significantly prevented by prior addition of Mg2+, suggesting direct effects of Mg2+ on the mitochondria. Moreover, Mg2+ supplementation also suppressed H2O2-induced cell death. In summary, this study revealed that H2O2-induced Mg2+ dissociation from ATP and that the resulting increase in [Mg2+]cyto prevented MMP depolarization in keratinocytes. Our results suggest that Mg2+ that has dissociated from ATP is not merely a byproduct, but functions as a cytoprotective mechanism against oxidative stress and that Mg2+ supplementation is effective in protection against oxidative stress.
Mg2+ mobilization from intracellular storage in keratinocytes has not yet been thoroughly investigated, while Mg2+ influx via NIPAL4, an Mg2+ transporter, was reported previously34,35,36. In the present study, we demonstrated that H2O2 increases cytosolic free Mg2+ by dissociation from ATP due to a decrease in cellular ATP level, although neither Mg2+ influx, Mg2+ transport via Na+/Mg2+ exchanger nor Mg2+ release from the mitochondria was involved in this response. It has been reported that mitochondria are intracellular Mg2+ storage sites and that depolarization of MMP induces Mg2+ release from the mitochondria into the cytosol in other cell types16,19,29,30. In contrast, [Mg2+]cyto instead decreased in response to FCCP in keratinocytes. This finding suggests that mitochondria do not act as Mg2+ sources in keratinocytes, although they probably contain Mg2+ in their matrix, similarly to other cell types, since some enzymes in the mitochondria require Mg2+ for their activity37,38. ATP is known to be a major intracellular Mg2+ binding partner: hundreds of enzymes utilize ATP in the form of Mg-ATP, and Mg2+ is dissociated upon the degradation of ATP into ADP, increasing intracellular free Mg2+13,32,39. Therefore, inhibition of mitochondrial ATP synthesis enhanced not only the decrease in ATP level but also the increase in [Mg2+]cyto that was induced by H2O2. In contrast, newborn keratinocytes, which showed relatively smaller decreases in ATP compared to adult keratinocytes, had smaller Mg2+ responses to H2O2 than adult keratinocytes. In the process of aging, the contribution of anaerobic respiration to the energy metabolism becomes substantial in keratinocytes40. This difference in the ATP production process between adult and newborn keratinocytes may cause larger ATP decreases and result in greater increases in Mg2+ in response to H2O2 in adult keratinocytes compared to newborn keratinocytes. Our results show that keratinocytes that exhibited large decreases in ATP showed large Mg2+ reactions in response to H2O2 and vice versa, strongly indicating that a major source of Mg2+ under oxidative stress conditions is the dissociation of Mg2+ from ATP.
In keratinocytes, oxidative stress changes metabolism drastically and acutely. Glucose usage changes from glycolysis to the pentose phosphate pathway within seconds of oxidative stress and decreased ATP levels occurrence11. Our study also focused on the acute response of keratinocytes to oxidative stress and demonstrated that reduced ATP levels lead to an increase in Mg2+ levels. Mg2+ has large effects on the cellular metabolism and also on mitochondrial functions. The positive effect of Mg2+ on MMP under mitochondrial stress conditions has been demonstrated not only in isolated mitochondria but also in cells20,21. The contributions of Mg2+ on MMP retention via inhibiting K+/H+ exchangers, preventing mitochondrial permeability transition pores (mPTP) from opening, and activating the tricarboxylic acid (TCA) cycle, has been reported previously41. In the present study, MMP levels under oxidative stress were also determined by [Mg2+]cyto, which was perturbed by oxidative stress. This result suggests that the change in [Mg2+]cyto is a mitochondria-protective mechanism under oxidative stress conditions. Previous studies have also demonstrated the long-term effects of Mg2+ on cell protection in keratinocytes42 and other cells22,24. [Mg2+]cyto changes the activity of phosphatases, such as mTOR, CREB, and ERK16, and leads to cell protection under stress conditions in keratinocytes42 and neurons22. Therefore, Mg2+ has both acute and long-term protective effects for cells.
Dissociation from ATP is a well-known source of Mg2+. However, little has previously been known about the role of the dissociated Mg2+. It has been reported that an increase in [Mg2+]cyto, which is probably dissociated from Mg-ATP, is required for DNA condensation during mitosis in HeLa cells18, indicating that increased [Mg2+]cyto resulting from ATP consumption is not a byproduct but instead plays an important role in cellular events. In the present study, we demonstrated that increasing levels of Mg2+ during the process of ATP level decrease protected the mitochondria, an organelle that is responsible for ATP generation, under oxidative stress in keratinocytes. This suggest that Mg2+ acts as a negative feedback signal to maintain ATP level against stress on cells. Some studies have already demonstrated that mitochondria was protected from oxidative stress under Mg2+ rich conditions, but these studies have not referred to changes in [Mg2+]cyto levels24,27,42. Our data reveal that [Mg2+]cyto increases in response to oxidative stress and that Mg2+ that dissociated from Mg-ATP is not a byproduct but instead acts as a mechanism to protect the mitochondria. Since the protective effect of Mg2+ supplementation against oxidative stress has been reported in other cell types23,24,25,26,27, the mechanism revealed here might be common in mammalian cells to protect cells against cellular stress.
In conclusion, we demonstrated that H2O2 induced an increase in [Mg2+]cyto due to dissociation from Mg-ATP, and the increased [Mg2+]cyto protected mitochondria from ROS damage. Moreover, supplementation of Mg2+ to extracellular medium further suppressed the decrease in MMP and attenuated H2O2 toxicity. The skin is always exposed to oxidative stress from UV, so Mg2+ would play an important role in maintaining the robustness of energy metabolism and protecting the skin from oxidative stress. Moreover, the addition of Mg2+ from an external source showed an additive effect for cell protection, suggesting that Mg2+ is a candidate active ingredient to protect skin from oxidative stress.
Normal human epidermal keratinocytes were purchased from Kurabo (Osaka, Japan). To compare the response of keratinocytes from newborn babies and adults, four different batches of newborn keratinocytes vials from different donors (0 years–1, 2, 3 and 4) and two different batches of adult keratinocytes vials (donors aged 40 years and 57 years) were used (details are summarized in Supplementary Table 1). Cells were cultured in EPILIFETM medium (Thermo Fisher Scientific, Waltham, MA, USA) supplemented with insulin (10 μg mL−1), human recombinant epidermal growth factor (0.1 ng mL−1), hydrocortisone (0.67 μg mL−1), gentamicin (50 μg mL−1), amphotericin B (50 ng mL−1), and bovine pituitary extract (0.4%, v/v), all of which were sourced from Kurabo, at 37 °C in a CO2 incubator. Undifferentiated keratinocytes between passage 2 and passage 6 were used for experiments. EPILIFETM medium contains Mg2+, but the concentration is not disclosed.
For fluorescence imaging, keratinocytes were seeded on glass bottom dishes (IWAKI, Shizuoka, Japan) coated with 5 μg mL−1 collagen (Sigma-Aldrich, Saint Louis, MO, USA) at a concentration of 6–8 × 104 cells mL−1.
Dye loading and fluorescence imaging
For Mg2+ imaging, cells were stained with an Mg2+-selective fluorescent probe, KMG-10443. Keratinocytes were incubated with 20 μM KMG-104-AM and 200 μg mL−1 Pluronic F-127 (Thermo Fisher Scientific) at 37 °C. After 30 min, the keratinocytes were washed twice with Ca2+-free HBSS (Thermo Fisher Scientific; the pH was buffered using 10 mM HEPES and adjusted to 7.4 with NaOH, HBSS contains 0.9 mM Mg2+) and incubated for a further 15 min in Ca2+-free HBSS to allow the complete hydrolysis of acetoxy methyl (AM) groups. To avoid differentiation of keratinocytes, all experimental procedures were performed in Ca2+-free medium.
For simultaneous imaging of cytosolic Mg2+ and MMP, keratinocytes that had been loaded with KMG-104 were then incubated in Ca2+-free HBSS containing 25 nM TMRE (Thermo Fisher Scientific) for 15 min at 37 °C. Fluorescence imaging was performed in Ca2+-free HBSS with 2.5 nM TMRE.
A confocal laser scanning microscope system, FluoView FV1000 (Olympus, Tokyo, Japan), was used for the measurement of fluorescence. For the imaging of Mg2+ alone, KMG-104 was excited at 488 nm using an Ar laser through a dichroic mirror (DM405/488, Olympus), and fluorescence at 510–610 nm was detected with a photomultiplier. Images were acquired every 4–6 s. For simultaneous imaging of cytosolic Mg2+ and MMP, KMG-104 and TMRE were simultaneously excited at 488 nm using an Ar laser and 559 nm from a laser diode, respectively, through a dichroic mirror (DM405/488/559, Olympus). The emitted fluorescence was separated at 560 nm (SDM560, Olympus) and observed at 505–545 nm for KMG-104 and 570–670 nm for TMRE.
Fluorescence imaging of intracellular ATP
A fluorescence resonance energy transfer (FRET)-type ATP sensor, ATeam1.0333, was kindly gifted from Dr. Imamura and was used to measure intracellular ATP levels. The plasmids that encoded ATeam were transfected into keratinocytes using Lipofectamine LTX (Thermo Fisher Scientific). These keratinocytes were cultured for 1–2 days after transfection to express the sensor proteins. Before observation, the cells were rinsed with and placed in Ca2+-free HBSS.
The cells were observed on the confocal laser scanning microscope system Fluoview FV1000. ATeam was excited at 440 nm using a laser diode through a dichroic mirror (DM405–440/515, Olympus), and the emitted fluorescence was separated by a dichroic mirror (SDM515, Olympus) and observed at 460–500 nm for CFP and at 515–615 nm for YFP.
For the negative control experiments, an ATP-insensitive variant of ATeam was constructed by inducing mutations of R122K and R126K in the ATP-sensing domain, following previous work33. The ATP-insensitive ATeam was expressed in the keratinocytes, and it was confirmed that H2O2 at a concentration below 10 mM had no impact on the fluorescent proteins.
The acquired images were analyzed using the software packages FluoView (Olympus), Aquacosmos (Hamamatsu Photonics, Shizuoka, Japan), and ImageJ. A region of interest (ROI) was assigned to the whole cell body of each cell, and the average fluorescence intensity in each ROI was calculated respectively. After subtracting the background, the time-course of fluorescence intensity for each cell (F) was normalized by the initial value (F0), and the resulting F/F0 values were compared between KMG-104 and TMRE. For ATeam, the ratio (R) of the fluorescence of cyan and yellow fluorescent protein (YFP/CFP) was calculated after subtracting background. The time-course of R was normalized by the initial value (R0), and the resulting R/R0 values were compared.
Two groups of data were compared using Student’s t-test. To compare multiple data sets, Dunnett’s test or Tukey’s tests were used. P < 0.05 was used to indicate significant differences.
Measurement of H2O2 sensitivity of KMG-104 in vitro
KMG-104 and several concentrations of H2O2 were mixed in 96-well plate and the fluorescence of these mixtures was measured using a Varioskan Flash spectral scanning multimode reader (Thermo Fisher Scientific). KMG-104 was excited at 500 nm and fluorescence intensity at 530 nm was measured and compared. H2O2 in the concentration range of 0–100 mM had no effect on the fluorescence of KMG-104 (Supplementary Fig. 1a).
Keratinocytes were plated at a density of 8.0 × 103 cells par well in a 96-well plate and incubated at 37 °C for more than 24 h. Medium was replaced to Mg2+ normal medium (normal EPILIFE with supplements) or Mg2+ +5 mM medium (EPILIFE with additional 5 mM Mg2+ and supplements) 10 min prior to H2O2 application. The medium was replaced to Mg2+ normal medium or Mg2+ +5 mM medium containing 0 or 1 mM H2O2, and cells were incubated for 24 h in the incubator. Then, the medium was replaced to 0.5 mg mL−1 MTT containing culture medium, and the cells were incubated for 2 h. The medium was discarded, and DMSO was then added to each well to dissolve the precipitate. The absorption at 575 nm was measured on a microplate reader, Valioscan (Thermo Fisher Scientific). The viabilities were calculated as a ratio to the average of H2O2 0 mM condition for each Mg2+ concentration.
Statistics and reproducibility
Fluorescence imaging experiments were repeated for 3–4 times for each experiment, and response of all the cells emitting sensor fluorescence in the field of view were analyzed. MTT assay was repeated for six times. Student’s t-test was used for comparison of a pair of data. Dannett’s test was used to compare multiple groups to control group. Tukey’s test was used to compare all differences among data group more than three groups.
Further information on research design is available in the Nature Portfolio Reporting Summary linked to this article.
All data used in this study have been uploaded as a supplementary file named “Supplementary data 1”.
FluoView (Olympus), Aquacosmos (Hamamatsu Photonics, Shizuoka, Japan), and ImageJ were used for process and analyze image data.
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The plasmid encoding ATeam was kindly gifted from Dr. Imamura (Kyoto University).
The authors declare no competing interests.
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Fujita, K., Shindo, Y., Katsuta, Y. et al. Intracellular Mg2+ protects mitochondria from oxidative stress in human keratinocytes. Commun Biol 6, 868 (2023). https://doi.org/10.1038/s42003-023-05247-6