Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy

It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophin-deficient mdx mice and patients with Duchenne muscular dystrophy (DMD). Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions. Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles. Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice. DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD. These downregulated genes are known as targets of FoxOs and TFEB. Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice. Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice. Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB. Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.


Transcriptional dysregulation of autophagy in the muscle of a mouse model of Duchenne muscular dystrophy
Ryuta Nakashima 1 , Ryusuke Hosoda 1 , Yuki Tatekoshi 1 , Naotoshi Iwahara 1,2 , Yukika Saga 1 & Atsushi Kuno 1* It has been reported that autophagic activity is disturbed in the skeletal muscles of dystrophindeficient mdx mice and patients with Duchenne muscular dystrophy (DMD).Transcriptional regulations of autophagy by FoxO transcription factors (FoxOs) and transcription factor EB (TFEB) play critical roles in adaptation to cellular stress conditions.Here, we investigated whether autophagic activity is dysregulated at the transcription level in dystrophin-deficient muscles.Expression levels of autophagy-related genes were globally decreased in tibialis anterior and soleus muscles of mdx mice compared with those of wild-type mice.DNA microarray data from the NCBI database also showed that genes related to autophagy were globally downregulated in muscles from patients with DMD.These downregulated genes are known as targets of FoxOs and TFEB.Immunostaining showed that nuclear localization of FoxO1 and FoxO3a was decreased in mdx mice.Western blot analyses demonstrated increases in phosphorylation levels of FoxO1 and FoxO3a in mdx mice.Nuclear localization of TFEB was also reduced in mdx mice, which was associated with elevated phosphorylation levels of TFEB.Collectively, the results suggest that autophagy is disturbed in dystrophin-deficient muscles via transcriptional downregulation due to phosphorylation-mediated suppression of FoxOs and TFEB.
Duchenne muscular dystrophy (DMD), which is caused by mutation of DMD gene encoding dystrophin, is an X-linked disease with poor clinical outcomes 1 .DMD is characterized by degeneration, necrosis, and skeletal muscle regeneration, resulting in progressive muscle weakness and atrophy.The general treatment of DMD is the use of a corticosteroid.Recently, exon skipping has been used as a new therapy for patients with DMD.However, exon skipping has limitations such as limited delivery of the drug to targeted tissues after administration and the inability to address all pathogenic mutations in the gene 2 .Therefore, there is a need to develop novel therapies based on the understanding of molecular mechanisms of the disease.
Autophagy is an intracellular system to degrade damaged proteins and organelles for recycling and is essential for cell survival 3 .In skeletal muscles, autophagy plays a critical role in the maintenance of muscle mass.Musclespecific deletion of Atg7, an essential autophagy effector gene, causes muscle atrophy 4 .Overexpression of Atg5, a gene involved in phagophore elongation, improves the age-related decline in muscle strength of mice 5 .The process of autophagy is controlled by nutrition-sensitive signals.It has been reported that mammalian target of rapamycin complex 1 (mTORC1), activated in a rich-nutrient condition 6 , inhibits autophagosome formation by phosphorylating ULK1 7,8 , Atg13 8 , and Atg14L 9 .mTORC1 also negatively regulates a step of autophagosome-lysosome fusion via phosphorylation of UVRAG 10 .AMPK has been shown to be a positive regulator of autophagosome formation through phosphorylation of ULK1 7 .Autophagy is also regulated at the transcriptional level.FoxO transcription factors (FoxOs) have been reported to positively regulate the expression of autophagy-related genes [11][12][13][14] .Phosphorylation of FoxOs by Akt promotes their nuclear export, thereby inhibiting their transcriptional activity 15,16 .Transcription factor EB (TFEB) also promotes the expression of autophagy-related and lysosomal genes [17][18][19] .Transcriptional activity of TFEB is also controlled by its phosphorylation status.Phosphorylation of TFEB by mTORC1 20,21 promotes its nuclear export and suppresses its transcriptional activity, whereas TFEB is dephosphorylated via calcineurin activated by MCOLN1-mediated calcium signaling 22 .
It has been reported that autophagic activity in the skeletal muscle is reduced in DMD patients and in mdx mice, a model of DMD [23][24][25] .De Palma et al. 23 and we 24 reported that the Akt-mTORC1 signal is enhanced in skeletal muscles from DMD patients and mdx mice.Restoration of autophagy by treatment of mdx mice with rapamycin, an inhibitor of mTORC1, led to an increase in muscle strength and an improvement of cardiac function 26 .These findings suggest that an enhanced Akt-mTORC1 signal is responsible for impaired autophagy and muscular and cardiac damage in DMD.In addition, it has been reported that the expression levels of autophagy-related genes are decreased in quadriceps, diaphragm, and tibialis anterior muscles in mdx mice 23,24 .We reported that treatment of mdx mice with resveratrol, an activator of the NAD + -dependent protein deacetylase SIRT1 27 , restores the expression of autophagy-related genes and ameliorates skeletal muscle injury 24,28 .Activation of TFEB via MCOLN1 has been reported to attenuate muscle damage in mdx mice 29 .These reports suggest that repression of autophagy-related genes contributes to suppressed autophagic activity and muscle injury in mdx mice.However, it remains unknown how autophagy-related genes are downregulated in DMD.Although it has been reported that nucleus-localized TEFB is reduced in the diaphragm of mdx mice 30 , it is unknown whether its transcriptional activity is downregulated via mTORC1-mediated phosphorylation in mdx mice.
In this study, we investigated whether transcription of autophagy-related genes is dysregulated in the skeletal muscle of mdx mice.We focused on FoxOs and TFEB, because Akt-mTORC1 signaling, which leads to suppression of those transcription factors, is activated in dystrophic muscles.

Autophagic activity is reduced in the skeletal muscle of mdx mice
We assessed the muscle strength of forearms in C57BL/10Sn mice as control wild-type (WT) and mdx mice at 12-13, 21-22, and 60-61 weeks of ages.As previously reported 31 , grip grasping power was lower in mdx mice than that in WT mice at those ages (Table 1).Because fast type fibers are firstly damaged in DMD patients 32 , we mainly analyzed tibialis anterior muscles at 22 weeks of age in this study.Western blotting showed that the protein level of LC3-II, an autophagosome marker, was significantly higher in the tibialis anterior muscle of mdx mice than in that of WT mice (Fig. 1a and b).The level of p62 protein, which is degraded by autophagy, was also higher in mdx mice (Fig. 1c and d).These data suggest that autophagosomes were accumulated via impaired autophagosome degradation in mdx mice.
We also examined the expression levels of autophagy-related genes in the soleus muscle, which mainly contains a slow type of muscle fibers, in mice at 22 weeks of age.We found that many autophagy-related genes were downregulated in the soleus muscle of mdx mice as observed in the tibialis anterior muscle (Supplementary Fig. S1).
Furthermore, we also analyzed autophagy-related genes in tibialis anterior muscles of mice at a younger age of 12 weeks (Supplementary Fig. S2).The expression levels of Map1lc3b, Becn1, Uvrag, Bnip3, Lamp1, and Fbxo32 were lower in mdx mice, suggesting suppressed activities of FoxOs and TFEB even at 12 weeks of age.www.nature.com/scientificreports/

FoxOs localized in the nucleus are reduced in mdx mice
Nuclear localization is essential for transcriptional activities of FoxOs.We therefore examined the localization of FoxO1 and FoxO3a in tibialis anterior muscles (Fig. 3a and c).Immunofluorescence showed that the percentage of FoxO1-positive nuclei was decreased in mdx mice compared with that in WT mice (Fig. 3a and b).FoxO3a localized in the nucleus was also reduced in mdx mice (Fig. 3c and d).These data collectively suggest that transcriptional activities of FoxO1 and FoxO3a were decreased in mdx mice.

Phosphorylation levels of FoxOs are increased in mdx mice
It has been reported that FoxOs are phosphorylated by Akt, and this phosphorylation leads to nuclear export of FoxOs and results in inhibition of their transcriptional activities 15,16 .Additionally, Akt has been reported to be activated in DMD patients and mdx mice 23 .We therefore investigated phosphorylation levels of FoxO1 and FoxO3a.Western blotting showed that phosphorylation levels of FoxO1 at Thr24 and FoxO3a at Thr32, phosphorylation sites by Akt 15,16 were increased in mdx mice (Fig. 4a-c).Levels of both phospho-Ser473-Akt and total Akt were increased in mdx mice, although the increase in the phospho-Akt level did not reach statistical significance (Fig. 4d-g).These results suggest that nuclear localization of FoxO1 and FoxO3a was decreased via their increased phosphorylation in mdx mice.
To further investigate the involvement of phosphorylation of FoxOs, we analyzed mdx mice at 71 weeks of age.Phosphorylation levels of FoxO1 and FoxO3a were also increased in mdx mice at this age (Supplementary Fig. S3c-e).These changes were associated with the increase in LC3-II levels in mdx mice (Supplementary Fig. S3a and b).Collectively, these data strongly indicate critical roles of FoxO phosphorylation on the regulation of autophagy.

Nuclear localization of TFEB is attenuated in mdx mice
We next performed immunofluorescence for TFEB with tibialis anterior muscles to investigate its intracellular localization in mdx mice.As expected, the number of TFEB-positive nuclei was significantly reduced in the tibialis anterior muscle of mdx mice (Fig. 5a and b), suggesting the reduction in TFEB activity in mdx mice.

Phosphorylation levels of TFEB are increased in mdx mice
It has been reported that TFEB is phosphorylated by mTORC1, leading to its nuclear export and transcriptional inactivation 20,21 .Moreover, mTORC1 activity has been reported to be increased in mdx mice 23,24 .Based on the knowledge, we hypothesized that phosphorylation of TFEB is increased in mdx mice.The phosphorylation level of TFEB at Ser122, one of the phosphorylation targets of mTORC1 21 , was increased in mdx mice (Fig. 6a and b).As expected, phosphorylation level of S6 ribosomal protein, a downstream effector of mTORC1, at Ser240/244 was higher in mdx mice than in WT mice (Fig. 6c and d).The protein level of calcineurin A, which is the catalytic subunit of calcineurin known to dephosphorylate TFEB 22 , was not reduced in mdx mice (Fig. 6e and f).At 71 weeks of age, phospho-TFEB levels were also higher in mdx mice (Supplementary Fig. 3f and g).Protein levels of calcineurin A were comparable in WT and mdx mice (Supplementary Fig. S3f and h).These data suggest that TFEB activity was decreased in mdx mice via mTORC1-mediated phosphorylation and subsequent nuclear export.

Resveratrol treatment restores autophagic activity and reduces phospho-TFEB level in tibialis anterior muscles of mdx mice
We previously reported that treatment of mdx mice with resveratrol, a known activator of the NAD + -dependent SIRT1, increases autophagic activity and expression levels of autophagy-related genes, including those that are targets of FoxOs and TFEB, in quadriceps muscles 24 .Additionally, in our earlier research, we observed an increase in the nuclear localization of FOXO3a as a result of resveratrol treatment in the hearts of mdx mice 47 .Furthermore, it has been documented that resveratrol treatment can lead to dephosphorylation of TFEB and its nuclear translocation in non-myocytes 48 .Therefore, we examined whether resveratrol treatment changes the phosphorylation status of FoxOs and TFEB in tibialis anterior muscles of mdx mice.Administration of resveratrol (0.4 g/kg food) to mdx mice was started at 9 weeks of age, and the muscle tissues were examined at 65 weeks of age as previously reported 24 .As observed in quadriceps muscles in our earlier study 24 , total LC3 (LC3-I + LC3-II) levels were reduced by resveratrol treatment, suggesting a restoration of autophagic activity (Supplementary Fig. S4a and b).Phosphorylation levels of FoxO1 and FoxO3 were not changed by resveratrol treatment (Supplementary Fig. S4c and d).On the other hand, phospho-TFEB levels were decreased by resveratrol treatment  www.nature.com/scientificreports/(Supplementary Fig. S4e and f).Resveratrol treatment did not change calcineurin A protein levels (Supplementary Fig. S4e and f).

Discussion
The present study showed that genes related to the autophagic process were globally downregulated in muscles of mdx mice.Nuclear FoxO1 and FoxO3a levels were decreased in mdx mice, which were associated with increases in phosphorylation levels of FoxO1 and FoxO3a.In addition, phospho-TFEB level was increased and nuclear TFEB was decreased in mdx mice.These findings suggest downregulation of autophagy-related genes in muscles of mdx mice via phosphorylation-mediated reduction in activities of FoxOs and TFEB.Downregulation of these genes was also observed in muscles from patients with DMD.This is the first report showing transcriptional dysregulation of autophagy in dystrophin-deficient skeletal muscles.The process of autophagy is separated into autophagosome formation, fusion of autophagosomes with lysosomes, and degradation of the contents of autophagosomes by lysosomal enzymes.In this study, we showed that levels of LC3-II, a marker of autophagosomes, were increased in the tibialis anterior muscle of mdx mice.Although transcript levels of p62 (Sqstm1) were similar in WT mice and mdx mice, p62 protein level was higher in mdx mice.These findings are consistent with results for the quadriceps muscle of mdx mice in our previous study 24 and in the diaphragm in a study by Spaulding HR et al. 30 .Although genes related to autophagosome formation were also downregulated, suppression of autophagosome clearance was dominantly involved in the impaired autophagic activity in mdx mice because the autophagosome marker LC3-II were accumulated in Western blotting analysis.Since lysosomal genes (Lamp1 and Atp6v1a) were not changed, the reduction in the expression levels of gene-related to autophagosome-lysosome fusion may have contributed to the suppression of autophagosome clearance in mdx mice.In addition, it is possible that activated mTORC1 also inhibits autophagosome-lysosome fusion by phosphorylating UVRAG 10 and VAMP8 49 .
Here, we report for the first time that transcriptional activities of FoxOs are reduced probably via their enhanced phosphorylation in mdx mice.Although Akt, which is known to phosphorylate FoxOs 15,16 , has been reported to be activated in skeletal muscle of mdx mice 23 , the increase in phospho-Akt levels in mdx mice did not reach statistical significance in the present study.Therefore, we cannot exclude the possibility that another kinase is also responsible for phosphorylation of FoxOs.Serum-and glucocorticoid-inducible kinase (SGK) phosphorylates FoxOs at the same sites as Akt 50 .There has been no study in which SGK activity in skeletal muscle of mdx mice was investigated.However, since mdx mice that lack SGK1 showed favorable phenotypes of skeletal muscles 51 , SGK may be activated in mdx mice.The other possible regulation of intracellular localization of FoxOs is mediated by SIRT1.FoxOs are deacetylation targets of SIRT1 52 and it has been reported that SIRT1 promotes nuclear localization of FoxOs 53,54 .Our previous study showed that administration of resveratrol, an activator of SIRT1, to mdx mice upregulated the expression levels of FoxO-regulated genes in skeletal muscle and the heart 24,47 .Furthermore, resveratrol treatment increased nuclear FoxO3a level in the heart of mdx mice 47 .Therefore, reduced SIRT1 activity may also be involved in nuclear exclusion of FoxOs in mdx mice.
Although nuclear TFEB was reported to be decreased in mdx mice 30 , the mechanism has not been determined.We found that the increase in phosphorylation level of TFEB at Ser122, a phosphorylation site by mTORC1, was associated with reduction in nucleus-localized TFEB in mdx mice.Since the phosphorylation level of S6, a downstream effector of mTORC1, was also elevated in mdx mice, it is possible that mTORC1-mediated TFEB phosphorylation led to its nuclear exclusion.Calcineurin dephosphorylates TFEB at Ser142 and Ser211 and increases transcriptional activity of TFEB 22 .However, calcineurin activity was reported to be unchanged in the tibialis anterior muscle and rather increased in the diaphragm of mdx mice at the age of 5 months 55 .Indeed, in the present study, the protein level of calcineurin A was not reduced in mdx mice.Therefore, it is unlikely that reduced dephosphorylation by calcineurin is responsible for increased phosphorylation of TFEB in mdx mice.
Since FoxOs and TFEB share many genes as targets, it is difficult to determine which transcription factors are dominantly affected in dystrophin-deficient muscles.Genes related to lysosomes such as Lamp1 and Atp6v1a, which are main targets of TFEB, were not changed in the tibialis anterior muscles of mdx mice.Furthermore, we observed that expression levels of LAMP1, ATP6V1a, and MCOLN1 in muscles of DMD patients were unchanged or rather upregulated despite a reduction in the expression of TFEB.These findings suggest that FoxOs, rather than TFEB, might play a dominant role in the downregulation of autophagy-related genes in the tibialis anterior muscle of mdx mice.In contrast, in the present study, expression levels Tfeb and Lamp1 were downregulated in the soleus muscles.In our previous study, the expression level of Tfeb in the quadriceps of mdx mice was reduced by approximately 50% compared to that in control mice 24 .In addition, TFEB activity determined by using a protein-DNA array has been reported to be increased in the diaphragm muscle from 6-week-old mdx mice 56 .Therefore, these findings suggest that changes in TFEB expression and activity may vary depending on skeletal muscle types.
One limitation of this study is that there was no information regarding other transcription factors involved in the expression of autophagy-related genes.It has been reported that the transcription factors E2F1 and NF-kB regulate Bnip3 expression 57,58 .However, activation of NF-kB has been demonstrated in the muscle of mdx mice 56 .Signal transducer and activator of transcription 1 (STAT1) negatively regulates autophagy by repressing the Ulk1 gene in skeletal muscle 59 and STAT1 activity has been reported to be markedly augmented in the muscle of mdx mice 56 .However, since the Becn1 gene was not altered in the skeletal muscle of STAT1 knockout mice 59 , downregulation of Becn1 observed in mdx mice cannot be explained by increased STAT1 activity.It has been reported that expression of autophagy-related genes is also regulated by epigenetic mechanisms including histone modifications and non-coding RNAs.We did not examine the roles of such regulations in this study.
Another limitation is the absence of experimental results directly demonstrating that the downregulation of autophagy-related genes in the muscles of mdx mice is a result of phosphorylation-mediated reductions in www.nature.com/scientificreports/activities of FoxOs and TFEB.In our previously study, we found that treatment of mdx mice with the SIRT1 activator resveratrol upregulated autophagy-related genes in the muscle 24 .In the present study, resveratrol treatment resulted in a reduction in phospho-TFEB levels in muscles from mdx mice.Furthermore, in a previous study, we found that resveratrol treatment increased the localization of FoxO3a in the nucleus of the hearts of mdx mice 47 .These findings support the notion that restoration of the activities of FoxOs and TFEB contributes to the upregulation of autophagy-related genes in the muscles of mdx mice, although further experiments are necessary.
In conclusion, expression of autophagy-related genes is globally downregulated in dystrophin-deficient dystrophic muscles in mice and humans.This could result from suppression of activities of FoxOs and TFEB.It is expected that correction of dysregulated transcription of autophagy-related genes may restore autophagic activity and attenuate dystrophic changes in muscles, though further studies are needed.

Animals
Animal experiments were approved by the Animal Care and Use Committee of Sapporo Medical University (08-100_10-118_11-046, 18-056_21-024) were performed according to the Guide for the Care and Use of Laboratory Animals (Institute of Laboratory Animal Resources, 1996) and the Animal Care and Use Committee of Sapporo Medical University.All methods were reported in accordance with the ARRIVE guidelines.Male mdx mice (C57BL/10ScSn-Dmdmdx/Jic) were obtained from the Central Institute for Experimental Animals (Kanagawa, Japan).Age-matched control mice (C57BL/10SnSlc) were purchased from Japan SLC, Inc. (Shizuoka, Japan).The mice were sacrificed for obtaining skeletal muscle tissue samples at 12, 22, and 71 weeks of ages.Skeletal muscle tissues were flash-frozen in liquid nitrogen and stored at − 80 °C until use.
In the present study, we analyzed tibialis anterior muscles obtained from untreated and resveratrol-treated mdx mice which were used in our previously study 24 .Treatment with resveratrol (0.4 g/kg food) was initiated at 9 weeks of age, and muscle tissues were sampled at 65 weeks of age 24 .

Grip test
Forearm grip strength was evaluated by using a grip strength meter (GPM-100B, MELQUEST, Toyama, Japan).The same operator who was blinded to the genotype of the mice performed the tests.The tests were conducted at 12-13, 21-22, and 60-61 weeks of ages.We assessed grip strength power at 1-min intervals We obtained the mean value from three trials per mouse for analysis.

Analysis of gene expression by real-time quantitative PCR
Total RNA was isolated from tibialis anterior and soleus muscles using the RNeasy Fibrous Tissue Mini Kit (Qiagen, Hilden, Germany).Complementary DNA was generated from total RNA using the GoScript Reverse Transcription System (Promega, Madison, WI, USA).DNA amplification was performed by the StepOne Real-Time PCR System (Thermo Fisher Scientific, Waltham, MA, USA) using GoTaq qPCR Master Mix (A600A, Promega, Madison, WI, USA) and CXR Reference Dye (C541A, Promega, Madison, WI, USA).Each sample was run in duplicate, and the mean value was used to evaluate the mRNA levels.All data were normalized to 18 s ribosomal RNA.The primer sequences are listed in Supplementary Table S2.

Analysis of gene expression in skeletal muscle of patients with Duchenne muscular dystrophy from the NCBI database
Differences in gene expression in healthy controls and patients with DMD were analyzed by using microarray datasets downloaded from Gene Expression Omnibus (https:// www.ncbi.nlm.nih.gov/ geo/).The following datasets were analyzed: GSE1004_GPL8300 (controls vs. DMD patients, source: quadriceps), GSE1007_GPL92 (controls vs. DMD patients, source: quadriceps), GSE1007_GPL93 (controls vs. DMD patients, source: quadriceps), GSE1007_GPL94 (controls vs. DMD patients, source: quadriceps) and GSE1007_GPL95 (controls vs. DMD patients, source: quadriceps).We analyzed data for which the number of absent values was less than one third of the total number of samples.We compared the mean values between controls and DMD patients.In the present study, we did not use human tissues for analyses.

Figure 1 .
Figure 1.Autophagic activity is reduced and autophagy-related genes are downregulated in tibialis anterior muscles in mdx mice.(a) Representative Western blot images for LC3 and GAPDH in tibialis anterior muscles from WT mice and mdx mice at 22 weeks of age.kDa: kilodalton.(b) Quantitative data of LC3-II levels normalized to GAPDH (N = 4).(c) Representative Western blot images for p62 and GAPDH in tibialis anterior muscles from WT mice and mdx mice at 22 weeks of age.(d) Quantitative data of p62 levels normalized to GAPDH (N = 4).(e-h) Expression levels of genes related to autophagosome formation (e), the step of autophagosome-lysosome fusion (f), and mitophagy (g), and other target genes of FoxOs or TFEB (h) in tibialis anterior muscles of WT mice and mdx mice at 22 weeks of age (N = 4) examined by real-time quantitative PCR.Target genes of FoxOs and TFEB are indicated.All data are expressed as means ± standard deviation.All statistical tests were conducted by Welch's two sample t-test.a.u.= arbitrary unit.*P < 0.05, **P < 0.01; NS, not significant.

Figure 3 .
Figure 3. Nuclear localization of FoxO1 and FoxO3a is decreased in tibialis anterior muscles of mdx mice.(a) Representative images of immunofluorescence for FoxO1 (green) and laminin (red), nuclear staining with Hoechst33342 (blue) and merged images in tibialis anterior muscles of WT mice and mdx mice at 22 weeks of age.The arrow indicates FoxO1 co-localized with the nucleus (Hoechst33342).Scale bar: 50 μm.(b) Percentage of FoxO1-positive nuclei.We analyzed 15 randomly selected images from 3 mice (N = 15 in each group).(c) Representative images of immunofluorescence for FoxO3a (green) and laminin (red), Hoechst33342 staining (blue) and merged images in tibialis anterior muscle sections of WT mice and mdx mice.The arrow indicates FoxO3a localized in the nucleus.Scale bar: 50 μm.(d) Percentage of FoxO3a-positive nuclei.We analyzed 15 randomly selected images from 3 mice in each group (N = 15).All data are expressed as means ± standard deviation.All statistical tests were conducted by Welch's two sample t-test.*P < 0.05.

Figure 4 .
Figure 4. Phosphorylation levels of FoxO1 and FoxO3a are increased in tibialis anterior muscles of mdx mice.(a) Western blot images for phospho-FoxO1 (Thr24), phospho-FoxO3a (Thr32), FoxO1, FoxO3a and GAPDH in tibialis anterior muscles from WT mice and mdx mice at 22 weeks of age.kDa: kilodalton.(b) Quantitative data of phosphorylation levels of FoxO1 normalized to FoxO1 (N = 4).(c) Quantitative data of phosphorylation levels of FoxO3a normalized to FoxO3a (N = 4).(d) Western blot images for phospho-Akt (Ser473) and total Akt in WT and mdx mice.(e-g) Quantitative data of phosphorylation and total levels of Akt normalized to GAPDH (N = 4).All data are expressed as means ± standard deviation.All statistical tests were conducted by Welch's two sample t-test.a.u.= arbitrary unit.*P < 0.05, **P < 0.01; NS, not significant.

Figure 5 .
Figure 5. TFEB localized in the nucleus is reduced in tibialis anterior muscles in mdx mice.(a) Representative images of immunofluorescence staining for TFEB (green) and laminin (red), nuclear staining with Hoechst33342 (blue) and merged images in tibialis anterior muscles of WT mice and mdx mice at 22 weeks of age.The arrow indicates TFEB located in the nucleus.Scale bar: 50 μm.(b) Percentage of TFEB-positive nuclei.We analyzed 15 randomly selected images from 3 mice in each group (N = 15).All data are expressed as means ± standard deviation.All statistical tests were conducted by Welch's two sample t-test.*P < 0.05.

Figure 6 .
Figure 6.Phosphorylation levels of TFEB and S6 protein are increased in tibialis anterior muscles in mdx mice.(a) Representative Western blot images for phospho-TFEB (Ser122), TFEB and GAPDH in tibialis anterior muscles of WT mice and mdx mice at 22 weeks of age.kDa: kilodalton.(b) Quantitative data of phosphorylation levels of TFEB normalized to TFEB (N = 4).(c) Representative Western blot images for phospho-S6 ribosomal proteins (Ser240/244), S6 ribosomal protein and GAPDH in tibialis anterior muscles from WT mice and mdx mice at 22 weeks of age.(d) Quantitative data of phosphorylation levels of S6 ribosomal protein normalized to S6 ribosomal protein (N = 4).(e) Representative Western blot images for calcineurin A. (f) Quantitative data of calcineurin A normalized to GAPDH (N = 4).All data are expressed as means ± standard deviation.All statistical tests were conducted by Welch's two sample t-test.a.u.= arbitrary unit.*P < 0.05; NS, not significant. https://doi.org/10.1038/s41598-024-51746-9

Table 1 .
Grip strength and body weight in mdx mice.All data are expressed as means ± standard deviation.All statistical tests were conducted by Welch's two sample t-test.