GW(g)/BW(g) GW(g)/BW(g) Con Dex Con Dex. GW(g)/BW(g) Relative mrna levels. Atrogin-1 Murf-1. Atrogin-1 Murf-1. Soleus

Transcription

1 a b c GW(g) GW(g) GW(g) Den * ** GW(g)/BW(g) Den Dex Dex GW(g)/BW(g) d 0.00 Fasting GW(g) e 0.15 Cancer Cachexia f GW(g) Young ** Old g GW(g)/BW(g) Young Old Relative mir-29b level Atrogin-1 Murf-1 TA Atrogin-1 Murf-1 Soleus Atrogin-1 Murf-1 Supplementary Figure 1. Multiple types of muscle atrophy in vivo (a) Gastrocnemius muscle weight (GW) and gastrocnemius muscle weight/body weight (GW/BW) ratio were reduced in denervation (Den) mice compared to control () group (n=5 per group). (b) GW and GW/BW ratio were reduced in dexamethasone (Dex)-treated mice compared to control group (n=5 per group). (c) GW was reduced in fasting mice compared to control group (n=5 per group). (d) GW was reduced in cancer cachexia mice compared to control group (n=5 per group). (e) GW and GW/BW ratio were reduced in ageing (Old) mice compared to control (Young) mice (n=4 per group). (f) qrt-pcr analysis of mir-29b expression in gastrocnemius muscle from denervation rat at day 3, 5, 7, and 14, compared to controls (n=5 per group). (g) qrt-pcr analysis showed increased Atrogin-1, Murf-1, and mir-29b expressions in tibialis anterior (TA), soleus, and extensor digitorum longus (EDL) from denervation mice compared to controls (n=5 per group). Error bars, SEM. An unpaired, two-tailed Student s t test was used for comparisons between two groups (a-e, g). One-way ANOVA test was performed to compare multiple groups followed by Bonferroni's post hoc test (f). *, P<0.05. **, P<0.01. EDL Relative mir-29b level TA Soleus EDL

2 a TNF-α Relative diameter of myotube TNF-α Relative mir-29b level Atrogin-1 Murf-1 TNF-α b H 2 O 2 Relative diameter of myotube H 2 O 2 Relative mir-29b level Atrogin-1 Murf-1 H 2 O 2 Supplementary Figure 2. mir-29b is increased in muscle atrophy cell models (a) TNF-α (100 ng/ml) induced muscle atrophy in C2C12 myotubes as evidenced by reduced myotube diameter, accompanied with increased Atrogin-1, Murf-1, and mir-29b expression levels (n=4 per group, scale bar: 100 μm). (b) H 2 O 2 (400 μm) induced muscle atrophy in C2C12 myotubes as evidenced by reduced myotube diameter, accompanied with increased Atrogin-1, Murf-1, and mir-29b expression levels (n=4 per group, scale bar: 100 μm). Error bars, SEM. An unpaired, two-tailed Student s t test was used for comparisons between two groups. *, P<0.05. **, P<0.01.

3 a Relative mir-29b level Fugw Fugw-miR-29b b Fugw Fugw-miR-29b Relative diameter of myotube Fugw Fugw-miR-29b c Fugw Fugw-miR-29b Atrogin-1 * * Murf-1 Supplementary Figure 3. mir-29b is sufficient to induce muscle atrophy in vitro (a) qrt-pcr analysis showed increased mir-29b expression in C2C12 myotubes transfected with FugwmiR-29b compared to Fugw control (n=6 per group). (b) Immunofluorescent staining for C2C12 myotubes showed reduced myotube diameter when transfected with Fugw-miR-29b (n=4 per group, scale bar: 100 μm). (c) qrt-pcr analysis showed up-regulated Atrogin-1 and Murf-1 expressions in C2C12 myotubes transfected with Fugw-miR-29b (n=6 per group). Error bars, SEM. An unpaired, two-tailed Student s t test was used for comparisons between two groups. *, P<0.05. **, P<0.01.

4 a b CK(ng/mL) NC-i+TNF-α mir-29b-i+tnf-α NC-i+H 2 O 2 mir-29b-i+h 2 O 2 CK(ng/mL) Atrogin-1 Atrogin-1 Murf-1 Murf-1 Relative diameter of myotube Relative diameter of myotube Supplementary Figure 4. mir-29b is necessary for muscle atrophy in vitro (a) mir-29b inhibition attenuated TNF-α (100 ng/ml) induced muscle atrophy in C2C12 myotubes as determined by myotube diameter (n=4 per group, scale bar: 100 μm), creatine kinase (CK) activity (n=6 per group), and Atrogin-1 and Murf-1 expressions (n=6 per group). (b) mir-29b inhibition attenuated H 2 O 2 (400 μm) induced muscle atrophy in C2C12 myotubes as determined by myotube diameter (n=4 per group, scale bar: 100 μm), creatine kinase (CK) activity (n=6 per group), and Atrogin-1 and Murf-1 expressions (n=6 per group). Error bars, SEM. One-way ANOVA test was performed to compare multiple groups followed by Bonferroni's post hoc test. *, P<0.05. **, P<0.01.

5 a Dex TNF-α H 2 O 2 b Relative to Relative to Relative to * * H 2 O 2 Den Dex Fasting Relative to Relative to Relative to * ** Fasting Supplementary Figure 5. and are reduced in muscle atrophy (a) Western blot showed that and were down-regulated in dexamethasone (Dex)-, TNF-α-, and H 2 O 2 -induced muscle atrophy in vitro (n=3 per group). (b) Western blot showed that and were down-regulated in denervation (Den)-, Dex-, and fasting-induced muscle atrophy in vivo (n=3 per group). Error bars, SEM. The presented blots are representative samples of three independent experiments. An unpaired, two-tailed Student s t test was used for comparisons between two groups. *, P<0.05. **, P<0.01.

6 a (S473) T-AKT (S253) T-FOXO3A Den P-mTOR T-mTOR P-P70S6K T-P70S6K Den Relative protein levels b Dex Dex (S473) T-AKT (S253) T-FOXO3A P-mTOR T-mTOR P-P70S6K T-P70S6K Relative protein levels c (S473) T-AKT (S253) T-FOXO3A Fasting P-mTOR T-mTOR P-P70S6K T-P70S6K Fasting Relative protein levels Supplementary Figure 6. -AKT Signaling is decreased in muscle atrophy (a) Western blot showed that the -AKT signaling (AKT, FOXO3A, mtor, and P70S6K phosphorylation levels) was down-regulated in denervation (Den)-induced muscle atrophy mouse model (n=3 per group). (b) Western blot showed that the -AKT signaling was down-regulated in dexamethasone (Dex)-induced muscle atrophy mouse model (n=3 per group). (c)western blot showed that the -AKT signaling was down-regulated in fasting-induced muscle atrophy mouse model (n=3 per group). Error bars, SEM. An unpaired, two-tailed Student s t test was used for comparisons between two groups. *, P<0.05.

7 a b c NC agomir mir-29b agomir % Distribution typei typeiia typeiib d f Myod Myog Myf5 e g IL-1α IL-1β IL-6 h NC agomir mir-29b agomir Relative to PI3K(P85α) i ( S473) T-AKT (S253) T-FOXO3A NC agomir mir-29b agomir (S473)/AKT (S253)/FOXO3A P-mTOR T-mTOR P-P70S6K T-P70S6K NC agomir mir-29b agomir P-mTOR/mTOR P-P70S6K/P70S6K * Supplementary Figure 7. mir-29b is sufficient to induce muscle atrophy in vivo (a) Cross sections of gastrocnemius were stained with MHC antibodies to identify type I (blue), type IIA

8 (green), and type IIB (red) muscle fibers, and to quantify myofiber type distribution in NC agomir and mir- 29b agomir treated mice (n=3 per group, scale bar: 50 μm). (b-e) qrt-pcr analysis of other ubiquitin ligases-related genes (b), autophagy-related genes (c), degeneration/regeneration-related genes (d), and inflammation-related genes (e) in the gastrocnemius from mice treated with mir-29b agomir (n=5 per group). (f)qrt-pcr analysis showed up-regulated transcriptional markers of denervation (Musk, Achra, Achre, Achrg, Cpla2, Ncam, andrunx1) in the gastrocnemius from denervated (Den) mice compared to controls (n=5 per group). (g) qrt-pcr analysis showed up-regulated transcriptional markers of denervation (Achre and Cpla2) in the gastrocnemius from mice treated with mir-29b agomir (n=5 per group). (h) Western blot showed that and were down-regulated in mir-29b agomir treated mice (n=3 per group). (i)western blot showed that the -AKT signaling (AKT, FOXO3A, mtor, and P70S6K phosphorylation levels) was down-regulated in mir-29b agomir treated mice (n=3 per group). Error bars, SEM. An unpaired, two-tailed Student s t test was used for comparisons between two groups. *, P<0.05. **, P<0.01.

9 a PI3K(p85a)/ / b (S473) T-AKT (S253) T-FOXO3A (S473)/AKT (S253)/FOXO3A P-mTOR T-mTOR P-P70S6K T-P70S6K P-mTOR/mTOR P-P70S6K/P70S6K Supplementary Figure 8. mir-29b is necessary for muscle atrophy in vivo (a) Western blot analysis showed that and were up-regulated in the gastrocnemius from mice treated with mir-29b sponge compared to Fugw control in the presence or absence of denervation (Den) (n=3 per group). (b)western blot analysis showed increased phosphorylation levels of AKT, FOXO3A, mtor, and P70S6K in the gastrocnemius from mice treated with mir-29b sponge compared to Fugw control in the presence or absence of denervation (Den) (n=3 per group). Error bars, SEM. One-way ANOVA test was performed to compare multiple groups followed by Bonferroni's post hoc test.*, P<0.05.

10 a GW(g) d GW(g)/BW(g) b Atrogin-1 e Murf-1 c Relative mir-29b level Relative mir-29b level TA Soleus EDL Relative mir-29b levels f TA Soleus EDL Fugw+sham Fugw+Imo mir-29b sponge +sham mir-29b sponge +Imo g h Fugw+Sham Fugw+Imo i 85KD mir-29b sponge+sham mir-29b sponge+imo Feret Diameter Atrogin-1 Murf-1 / / GW(g) GW(g)/BW(g) Supplementary Figure 9. mir-29b in immobilization-induced muscle atrophy (a) Gastrocnemius muscle weight (GW) and Gastrocnemius muscle weight/body weight (GW/BW) ratio

11 were reduced in immobilization mice compared to control group (n=6 per group). (b) qrt-pcr showed that Atrogin-1 and Murf-1 were up-regulated in the gastrocnemius from immobilization mice (n=6 per group). (c) qrt-pcr showed that mir-29b was up-regulated in the gastrocnemius from immobilization mice (n=6 per group). (d) qrt-pcr analysis showed increased Atrogin-1, Murf-1, and mir-29b expressions in the tibialis anterior (TA), soleus, and extensor digitorum longus (EDL) from immobilization mice compared to controls (n=6 per group). (e) qrt-pcr showed that mir-29b sponge significantly decreased mir-29b expression in the gastrocnemius (n=5 per group). (f) Gastrocnemius muscle morphology, GW, and GW/BW ratio showed that mir-29b sponge at least partly blocked immobilization-induced muscle atrophy (n=5 per group, scale bar: 1 cm). (g) qrt-pcr analysis showed that Atrogin-1 and Murf-1 were no longer up-regulated in immobilization mice treated with mir-29b sponge (n=5 per group). (h) Hematoxylin-eosin (HE) staining showed that the reduction in muscle fiber diameter in immobilization mice was partly attenuated by mir- 29b sponge (n=5 per group, scale bar: 50 μm). (i) Western blot analysis showed that the down-regulation of and expression levels in immobilization mice were blocked by mir-29b sponge (n=3 per group). Age and sex matched mice were used for experiments randomly. Error bars, SEM. An unpaired, twotailed Student s t test was used for comparisons between two groups (a-d). One-way ANOVA test was performed to compare multiple groups followed by Bonferroni's post hoc test (e-i). *, P<0.05. **, P<0.01.

12 Supplementary Figure 10. Uncropped scans of Western blots Western blots for Fig.5b 26KD 26KD Western blots for Fig.5c Western blots for Fig.5d (T308) (T308) (S473) (S473) T-AKT T-AKT (S253) (T32) (S253) T-FOXO3A (T32) T-FOXO3A P-mTOR 250KD T-mTOR 250KD P-mTOR 315KD 250KD P-P70S6K T-mTOR 315KD 250KD T-P70S6K P-P70S6K P-4EBP1 T-P70S6K P-4EBP1 T-4EBP1 T-4EBP1

13 Western blots for Fig.7d Western blots for Fig.8i 130KD YY1 65KD Ubiquitin 43KD YY1 65KD Western blots for Fig.8k YY1 65KD Myosin/ MHC 250KD 180KD 227KD Western blots for Fig.8l P62 62KD LC3-I LC3-II 18KD 16KD

14 Western blots for Sup Fig.5a Western blots for Sup Fig.5b 26KD 26KD 26KD 26KD 26KD

15 Western blots for Sup Fig.6a (S473) P-mTOR 250KD T-AKT T-mTOR 250KD (S253) P-P70S6K T-P70S6K T-FOXO3A Western blots for Sup Fig.6b P-mTOR 250KD (S473) T-mTOR 250KD T-AKT P-P70S6K (S253) T-P70S6K T-FOXO3A

16 Western blots for Sup Fig.6c (S473) P-mTOR 315KD T-AKT T-mTOR 315KD (S253) P-P70S6K T-FOXO3A T-P70S6K Western blots for Sup Fig.7h 26KD

17 Western blots for Sup Fig.7i ( S473) P-mTOR 315KD T-AKT T-mTOR 315KD (S253) P-P70S6K T-FOXO3A T-P70S6K Western blots for Sup Fig.8a

18 Western blots for Sup Fig.8b (S473) P-mTOR 315KD 250KD T-AKT T-mTOR 315KD 250KD (S253) P-P70S6K T-FOXO3A T-P70S6K Western blots for Sup Fig.9i 85KD 26KD

19 Supplementary Table 1.Aberrant expressed micrornas in denervated muscle MicroRNA Regulation Fold change P value mir-130b up mir-21 up mir-29b up mir-212 up mir-672 up mir-223 up mir-222 up mir-221 up mir-203 up mir-132 up mir-511* up mir-146b up mir-133a* down mir-1949 up mir-326 up

20 Supplementary Table 2. sirna sequences used in this study Gene mmu-yy1 Sequence (5'-3') GCCCTCATAAAGGCTGCAC CCCTAAGCAACTGGCAGAA mmu- GCCTTCCAACTCAATTATT GCCTCTGTGACTTCTTGAA

21 Supplementary Table 3. Primer sequences for qrt-pcrs used in this study Gene Forward primer(5'-3') Reverse primer(5'-3') rno-atrogin-1 TTCCATCAGGAGAAGTGGATCT GGCAGTCGAGAAGTCCAGTC rno-murf-1 CACGAAGACGAGAAAATCAACA TGTCCTTGGAAGATGCTTTGTA rno-18s TCAAGAACGAAAGTCGGAGG GGACATCTAAGGGCATCAC mmu-mhc GAGGGTGGCTCTCACACATTC TTGGCCTTCGTAAGCAAACTG mmu-myod CCACTCCGGGACATAGACTTG AAAAGCGCAGGTCTGGTGAG mmu-myog GAGACATCCCCCTATTTCTACCA GCTCAGTCCGCTCATAGCC mmu-myf5 AAGGCTCCTGTATCCCCTCAC TGACCTTCTTCAGGCGTCTAC mmu-il-1α CGAAGACTACAGTTCTGCCATT GACGTTTCAGAGGTTCTCAGAG mmu-il-1β GCAACTGTTCCTGAACTCAACT ATCTTTTGGGGTCCGTCAACT mmu-il-6 TAGTCCTTCCTACCCCAATTTCC TTGGTCCTTAGCCACTCCTTC mmu-yy1 CAGTGGTTGAAGAGCAGATCAT AGGGAGTTTCTTGCCTGTCAT mmu-traf6 AAAGCGAGAGATTCTTTCCCTG ACTGGGGACAATTCACTAGAGC

22 mmu-nedd4 TCGGAGGACGAGGTATGGG GGTACGGATCAGCAGTGAACA mmu-mul1 CTGGGCACCAGTTCGATGG GACAGCATAAGGCACACACTT mmu-znf216 CCCATGCTGTGTAGTACAGGA GCTCATTCTGCCACTATTCTGC mmu-fbxo40 CGTCTCCTGCCTGGTGATAAG GTATGCTCTGACTCTTTGCACAT mmu-murf2 AAAGCAACTGATCTGTCCCATC TGTGGGTAAGTACGGGTTAGAG mmu-murf3 GGAGAAGCAGCTCATTTGCC CCTCCTGAAGACACCGTTGTG mmu-cblb GGTCGCATTTTGGGGATTATTGA TTTGGCACAGTCTTACCACTTT mmu-atg7 GTTCGCCCCCTTTAATAGTGC TGAACTCCAACGTCAAGCGG mmu-map1-lc3 CACTGCTCTGTCTTGTGTAGGTTG TCGTTGTGCCTTTATTAGTGCATC mmu-cathepsinl GTGGACTGTTCTCACGCTCAAG TCCGTCCTTCGCTTCATAGG mmu-gabarapl1 CATCGTGGAGAAGGCTCCTA ATACAGCTGGCCCATGGTAG mmu-bnip3 TTCCACTAGCACCTTCTGATGA GAACACCGCATTTACAGAACAA mmu-bnip3l TTGGGGCATTTTACTAACCTTG TGCAGGTGACTGGTGGTACTAA mmu-atg12 TCCGTGCCATCACATACACA TAAGACTGCTGTGGGGCTGA

23 mmu-vps34 TGTCAGATGAGGAGGCTGTG CCAGGCACGACGTAACTTCT mmu-beclin TGAATGAGGATGACAGTGAGCA CACCTGGTTCTCCACACTCTTG mmu-atg4b ATTGCTGTGGGGTTTTTCTG AACCCCAGGATTTTCAGAGG mmu-tbp TGTGAATACTGGTGCTGAG GGCATGAGACAAGACCTATA mmu-musk TTCAGCGGGACTGAGAAACT TGTCTTCCACGCTCAGAATG mmu-achra TCCCTTCGATGAGCAGAACT GGGCAGCAGGAGTAGAACAC mmu-achre GTGTCTGGATTGGCATTGACT ACACCTGCAAAATCGTCCTTG mmu-achrg GACCAACCTCATCTCCCTGA GAGAGCCACCTCGAAGACAC mmu-cpla2 GACAGCTCCGACAGTGATGA CGTCCTTCTCGGGTATTGAA mmu-ncam AAGGGGAAGGCACTGAATTT TCTCCTGCCACTTGACACAG mmu-runx1 AGCCTGGCAGTGTCAGAAGT TGGCATCTCTCATGAAGCAC mmu-myh1 GCGAATCGAGGCTCAGAACAA GTAGTTCCGCCTTCGGTCTTG mmu-myh2 AAGTGACTGTGAAAACAGAAGCA GCAGCCATTTGTAAGGGTTGAC mmu-myh4 CTTTGCTTACGTCAGTCAAGGT AGCGCCTGTGAGCTTGTAAA

24 mmu-myh7 ACTGTCAACACTAAGAGGGTCA TTGGATGATTTGATCTTCCAGGG muu-atrogin-1 CAGCTTCGTGAGCGACCTC GGCAGTCGAGAAGTCCAGTC Mmu- GTGGGGGCTCGTGTTTCTC GATCACCGTGCAGTTTTCCA mmu-murf-1 GTGTGAGGTGCCTACTTGCTC GCTCAGTCTTCTGTCCTTGGA mmu-18s TCAAGAACGAAAGTCGGAGG GGACATCTAAGGGCATCAC